UBC Theses and Dissertations

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UBC Theses and Dissertations

Membrane perturbational effects of inorganic cations Garnett, Maureen Elizabeth 1976

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MEMBRANE PERTURBATIONAL EFFECTS OF INORGANIC CATIONS by MAUREEN ELIZABETH GARNETT B.Sc., U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e D e p a r t m e n t i o f • • P h a r m a c o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1976 ^) Maureen E l i z a b e t h G a r n e t t , 1976 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I further agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thesis fo r f inanc ia l gain sha l l not be allowed without my written permission. Department of ^ H I M A c c - o &r The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date S ^ r i ABSTRACT The membrane p e r t u r b a t i o n a l e f f e c t s o f d i v a l e n t and m o n o v a l e n t c a t i o n s were examined by s t u d y i n g k i n e t i c and c h e m i c a l c h a r a c t e r i s t i c s o f t r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS) i n c o r p o r a t i o n i n t o t h e amino g r o u p s o f human e r y t h r o c y t e membranes. B o t h m o n o v a l e n t and d i v a l e n t c a t i o n s s t i m u l a t e d TNBS i n c o r p o r a t i o n . The m o n o v a l e n t c a t i o n -s t i m u l a t e d TNBS i n c o r p o r a t i o n , w h i c h was n o t s i z e d e p e n d e n t , was a t t r i b u t e d t o n o n - s p e c i f i c i o n i c s t r e n g t h e f f e c t s w h i l e d i v a l e n t c a t i o n - s t i m u l a t e d TNBS i n c o r p o r a t i o n , w h i c h was s i z e d e p e n d e n t , was e x p l a i n e d i n terms o f membrane s t r u c -t u r a l p e r t u r b a t i o n s w h i c h i n d i r e c t l y a f f e c t e d amino g r o u p r e a c t i v i t y . A p a r a l l e l was n o t e d between t h e e f f e c t s o f d i v a l e n t c a t i o n s on t h e a c t i v a t i o n e n e r g y f o r TNBS i n c o r -p o r a t i o n and t h e f i r s t i o n i z a t i o n p o t e n t i a l o f t h e c a t i o n s . I t was c o n c l u d e d t h a t t h e a c t i v a t i o n e n e r g y t e r m i s d e t e r -m ined p r i m a r i l y by e l e c t r o s t a t i c p r o p e r t i e s a f f e c t i n g p r o b e p e r m e a t i o n . L a b e l l i n g s t u d i e s i n d i c a t e d an i m p o r t a n t r o l e o f membrane p r o t e i n s i n m e d i a t i n g t h e s e e f f e c t s . S i m i l a r l y , p a r a l l e l s were a l s o n o t e d between c a t i o n - i n d u c e d r a t e e n -hancement o f TNBS i n c o r p o r a t i o n , a s s o c i a t e d w i t h a c o r r e s -p o n d i n g i n c r e a s e i n p h o s p h o l i p i d l a b e l l i n g by TNBS, and t h e s e c o n d i o n i z a t i o n p o t e n t i a l f o r t h e c a t i o n . T h i s was t h o u g h t t o i n d i c a t e t h a t t h e r a t e o f TNBS i n c o r p o r a t i o n was d e p e n d e n t p r i m a r i l y on p h o s p h o l i p i d s and t h a t d i v a l e n t c a t i o n s e x e r t e d t h e i r e f f e c t s on r a t e by a l t e r i n g t h e h y d r a t i o n o f p h o s p h o -l i p i d p o l a r h e a d g r o u p s , l e a d i n g t o a c h a n g e i n c o n f i g u r a -t i o n o f t h e p h o s p h o l i p i d s a n d h e n c e a c h a n g e i n t h e a c c e s s i -b i l i t y o f membrane amino g r o u p s . D i f f e r e n c e s w e r e f o u n d b e t w e e n t h e r e l a t i v e e x t e n t and t h e c h a r a c t e r i s t i c s o f p r o -t e i n a nd p h o s p h o l i p i d l a b e l l i n g b y TNBS i n t h e p r e s e n c e o f v a r i o u s c a t i o n s . B a s e d o n t h e r a t i o s o f p h o s p h o l i p i d t o p r o t e i n l a b e l l i n g , t h e d i v a l e n t c a t i o n s c o u l d be d i v i d e d i n t o two g r o u p s , a homogeneous g r o u p c o n s i s t i n g o f t h e 2+ 2+ 2+ 2+ a l k a l i n e e a r t h c a t i o n s Mg , Ca , S r , and Ba , and a s e c o n d h e t e r o g e n e o u s g r o u p i n c l u d i n g C o 2 + , N i 2 + , and M n 2 + . A s i m i l a r g r o u p i n g o f t h e s e i o n s c a n be made on t h e b a s i s o f t h e i r known e f f e c t s on t h e f u n c t i o n a l p r o p e r t i e s o f c e r -t a i n e x c i t a b l e t i s s u e s . S t u d i e s o f d i v a l e n t c a t i o n e f f e c t s on m e m b r a n e - a s s o c i a t e d enzymes i n d i c a t e d t h a t d i v a l e n t c a t i o n s c a n i n t e r a c t w i t h b o t h i n n e r and o u t e r membrane s u r f a c e s . The i n f l u e n c e o f d i v a l e n t a n d m o n o v a l e n t c a t i o n s o n u l t r a s o n i c d i s r u p t i o n o f membranes was e x a m i n e d i n a n o t h e r s e r i e s o f e x p e r i m e n t s . The r e s u l t s o b t a i n e d c l o s e l y p a r a l l e l e d t h o s e f o u n d w i t h TNBS i n c o r p o r a t i o n , i n d i c a t i n g t h a t c a t i o n e f f e c t s a n a l y z e d u s i n g TNBS as a p r o b e a r e a r e s u l t o f c a t i o n - m e m b r a n e i n t e r a c t i o n s , w i t h m i n i m a l c o n t r i -b u t i o n f r o m t h e p r o b e i t s e l f . F u r t h e r m o r e , i n e x p e r i m e n t s i n v o l v i n g a h y p o t o n i c c h a l l e n g e o f i n t a c t e r y t h r o c y t e s i n t h e p r e s e n c e o f d i v a l e n t o r m o n o v a l e n t c a t i o n s , t h e a n t i -h e m o l y t i c e f f e c t s o f t h e s e i o n s c l o s e l y f o l l o w e d t h e i r a b i l i t y t o s t i m u l a t e TNBS i n c o r p o r a t i o n i n t o membranes. F i n a l l y , p r e l i m i n a r y e x p e r i m e n t s w i t h b o v i n e s y n a p t i c v e s i c l e s and rat l i v e r microsomal membranes gave results q u a l i t a t i v e l y s i m i l a r to those obtained with erythrocytes, suggesting that information on the molecular mechanisms by which divalent cations perturb erythrocyte membranes may be applicable to other more complicated membrane systems. TABLE OF CONTENTS INTRODUCTION MATERIALS METHODS Membrane P r e p a r a t i o n s C o m p o s i t i o n a l Assays Enzyme Treatments Enzyme Assays M i s c e l l a n e o u s P r o c e dures RESULTS A c t i v a t i o n Energy and Rate S t u d i e s Role o f Membrane L i p i d s and P r o t e i n s Approach to F u r t h e r C h a r a c t e r i z a t i o n o f Ion-Induced P r o t e i n P e r t u r b a t i o n s E f f e c t s o f C a t i o n s on H e m o l y s i s o f I n t a c t E r y t h r o c y t e s T u r b i d i t y Measurements i n S o n i c a t e d Membrane Suspensions Ion-Induced P e r t u r b a t i o n s i n Other Membrane Systems DISCUSSION BIBLIOGRAPHY LIST OF TABLES T i t l e I n f l u e n c e o f C a t i o n s on R e l a t i v e L a b e l l i n g o f E r y t h r o c y t e Membrane P h o s p h o l i p i d s and P r o t e i n s by TNBS I n f l u e n c e o f C a t i o n s on R e l a t i v e L a b e l l i n g o f Bovine S y n a p t i c V e s i c l e and P u r i f i e d Rat L i v e r M i c r o s o m a l Membrane P h o s p h o l i p i d s and P r o t e i n s by TNBS L I S T OF FIGURES T i t l e A c t i v a t i o n E n e r g i e s f o r t h e I n c o r -p o r a t i o n o f TNBS and PC i n t o E r y t h r o -c y t e Membranes as a F u n c t i o n o f pH R a t e s o f I n c o r p o r a t i o n a t 25°C o f TNBS and PC i n t o E r y t h r o c y t e Membranes as a F u n c t i o n o f pH A c t i v a t i o n E n e r g i e s f o r t h e I n c o r -p o r a t i o n o f TNBS and PC i n t o B u t y l -amine as a F u n c t i o n o f pH R a t e s o f I n c o r p o r a t i o n a t 25°C o f TNBS and PC i n t o B u t y l a m i n e as a F u n c t i o n o f pH A r r h e n i u s P l o t s f o r t h e I n c o r p o r -a t i o n o f TNBS i n t o E r y t h r o c y t e Membranes i n t h e A b s e n c e o r P r e s e n c e o f M o n o v a l e n t o r D i v a l e n t C a t i o n s A c t i v a t i o n E n e r g i e s and R a t e s o f I n c o r p o r a t i o n a t 25°C f o r TNBS I n c o r -p o r a t i o n i n t o E r y t h r o c y t e Membranes i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s A c t i v a t i o n E n e r g i e s and R a t e s o f I n c o r p o r a t i o n a t 25°C f o r TNBS I n c o r -p o r a t i o n i n t o E r y t h r o c y t e Membranes i n t h e P r e s e n c e o f M o n o v a l e n t C a t i o n s T i t l e F i r s t and Second I o n i z a t i o n P o t e n t i a l s o f D i v a l e n t C a t i o n s A c t i v a t i o n E n e r g i e s and R a t e s o f I n c o r -p o r a t i o n a t 25°C f o r TNBS I n c o r p o r a t i o n i n t o U n t r e a t e d and N e u r a m i n i d a s e T r e a t e d E r y t h r o c y t e Membranes i n t h e A b s e n c e o r P r e s e n c e o f a D i v a l e n t o r M o n o v a l e n t C a t i o n A c t i v a t i o n E n e r g i e s and R a t e s o f I n c o r -p o r a t i o n a t 25°C f o r PC I n c o r p o r a t i o n i n t o E r y t h r o c y t e Membranes i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s E f f e c t s o f Enzyme P r e t r e a t m e n t o f E r y t h r o c y t e Membranes on D i v a l e n t C a t i o n -S t i m u l a t e d TNBS I n c o r p o r a t i o n TNBS L a b e l l i n g P a t t e r n s o f E r y t h r o c y t e Membranes i n t h e P r e s e n c e o r A b s e n c e o f D i v a l e n t C a t i o n s Maximal TNBS L a b e l l i n g o f P r o t e i n and P h o s p h o l i p i d Components o f E r y t h r o c y t e Membranes i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s M o d i f i c a t i o n o f E r y t h r o c y t e Membrane P h o s p h o l i p i d s by TNBS i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s T i t l e M o d i f i c a t i o n o f E r y t h r o c y t e Membrane S u l f h y d r y l Groups by DTNB i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s E f f e c t s o f D i v a l e n t C a t i o n s on A c e t y l c h o l i n e s t e r a s e A c t i v i t y E f f e c t s o f S u b s t i t u t i n g D i v a l e n t C a t i o n s f o r Mg + on t h e A c t i v i t y o f B a s a l and K + - S t i m u l a t e d p - N i t r o -p h e n y l p h o s p h a t a s e E f f e c t s o f D i v a l e n t C a t i o n s on B a s a l and K + - S t i m u l a t e d p - N i t r o p h e n y l p h o s -p h a t a s e A c t i v i t i e s E f f e c t s o f M o n o v a l e n t and D i v a l e n t C a t i o n s on H y p o t o n i c H e m o l y s i s o f I n t a c t E r y t h r o c y t e s E f f e c t s o f M o n o v a l e n t and D i v a l e n t C a t i o n s on U l t r a s o n i c D i s r u p t i o n o f E r y t h r o c y t e Membranes E f f e c t s o f D i v a l e n t C a t i o n s on t h e U l t r a s o n i c D i s r u p t i o n o f E r y t h r o c y t e Membranes E f f e c t s o f D i v a l e n t C a t i o n s on t h e U l t r a s o n i c D i s r u p t i o n o f E n z y m a t i c a l l y M o d i f i e d E r y t h r o c y t e Membranes T i t l e M aximal TNBS L a b e l l i n g o f P r o t e i n and P h o s p h o l i p i d Components o f B o v i n e Syn-a p t i c V e s i c l e s i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s Maximal TNBS L a b e l l i n g o f P r o t e i n and P h o s p h o l i p i d Components o f P u r i f i e d Rat L i v e r M i c r o s o m a l Membranes i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s E f f e c t s o f D i v a l e n t C a t i o n s on U l t r a -s o n i c D i s r u p t i o n o f R a t L i v e r M i c r o s o m e s , B o v i n e S y n a p t i c V e s i c l e s , and P u r i f i e d R a t L i v e r M i c r o s o m a l Membranes X ACKNOWLEDGEMENTS I would l i k e t o expr e s s the deepest a p p r e c i a t i o n and g r a t i t u d e t o Dr. D a v i d V. Godin f o r h i s enthusiasm, p a t i e n c e , and many hours o f v a l u a b l e d i s c u s s i o n d u r i n g the c o u r s e o f t h i s work. I would a l s o l i k e to thank Mrs. Therese Wan Ng f o r he r t e c h n i c a l a s s i s t a n c e . F i n a n c i a l s u p p o r t by the M e d i c a l Research C o u n c i l i s g r a t e f u l l y acknowledged. 1 INTRODUCTION A c o n s i d e r a b l e number o f e x p e r i m e n t a l s t u d i e s have p r o d u c e d e v i d e n c e as t o t h e e s s e n t i a l r o l e o f i n o r g a n i c c a t i o n s i n d e t e r m i n i n g b o t h s t r u c t u r a l and f u n c t i o n a l c h a r -a c t e r i s t i c s o f b i o l o g i c a l membranes. D e p l e t i o n o f d i v a -l e n t c a t i o n s i n r e d b l o o d c e l l s by t h e u s e o f c o m p l e x i n g a g e n t s o r by r e p l a c e m e n t w i t h o r g a n i c c a t i o n s l e a d s t o t h e s o l u b i l i z a t i o n o f a l a r g e p r o p o r t i o n o f t h e s t r u c t u r a l com-p o n e n t s o f t h e membrane and r e s u l t s i n t h e l o s s o f n o r m a l membrane m o r p h o l o g i c a l p r o p e r t i e s ( 1 ) . S t u d i e s w i t h b o v i n e e r y t h r o c y t e g h o s t s h a ve shown t h a t t h e s e c e l l s a r e more s u s c e p t i b l e t o p r o t e i n and l i p i d l o s s e s d u r i n g h y p o t o n i c w a s h i n g t h a n human e r y t h r o c y t e g h o s t s ( 2 ) . T h i s d e c r e a s e d s t a b i l i t y o f t h e b o v i n e e r y t h r o c y t e as com-p a r e d t o t h e human e r y t h r o c y t e i s b e l i e v e d t o be r e l a t e d t o d i f f e r e n c e s i n t h e d i v a l e n t c a t i o n b i n d i n g p r o p e r t i e s o f t h e s e c l o s e l y r e l a t e d membrane s y s t e m s . M o r e o v e r , t h e p r o -t e i n and l i p i d l o s s e s c a n be m a r k e d l y r e d u c e d i f t h e h y p o -t o n i c w a s h i n g i s p e r f o r m e d i n t h e p r e s e n c e o f d i v a l e n t 2+ 2+ 2+ 2 + c a t i o n s s u c h as Ca , Mg , Ba , o r S r . E l e c t r o n m i c r o -s c o p i c e x a m i n a t i o n o f t h e s e washed b o v i n e s t r o m a h a s shown 2 + t h a t t h e Mg - s t r o m a r e t a i n a l a r g e p o r t i o n o f t h e i r morpho-2 + l o g i c a l i n t e g r i t y and t h e Ca - s t r o m a r e m a i n m o r p h o l o g i c a l l y i n t a c t e v e n a f t e r e x t e n s i v e h y p o t o n i c w a s h i n g , w h i l e t h e c o n t r o l s t r o m a show a c o n s i d e r a b l e d e g r e e o f damage, a g a i n d e m o n s t r a t i n g t h e i m p o r t a n t r o l e o f d i v a l e n t c a t i o n s i n 2 m a i n t a i n i n g the s t r u c t u r a l i n t e g r i t y o f membranes. R e c o n s t i t u t i o n s t u d i e s o f membranes (3) have i n d i c a t e d t h a t p o l y v a l e n t c a t i o n s i n f l u e n c e the r e c o m b i n a t i o n o f s o l u -b i l i z e d membrane components. Membranes are g e n e r a l l y b e s t s o l u b i l i z e d by d e t e r g e n t s , which must be removed b e f o r e r e -c o m b i n a t i o n can o c c u r . T h i s can be done by a v a r i e t y o f methods, of which the most common ar e g e l f i l t r a t i o n and d i a l y s i s . Gel f i l t r a t i o n w i l l remove most of the d e t e r g e n t b u t d i v a l e n t c a t i o n s are n e c e s s a r y to r e c o n s t i t u t e the mem-brane and must be added a f t e r removal o f t h e d e t e r g e n t . However, d i r e c t a d d i t i o n o f d i v a l e n t c a t i o n s to s o l u b i l i z e d membrane components may cause p r e c i p i t a t i o n o f the components r a t h e r than membrane f o r m a t i o n . D i a l y s i s a g a i n s t a d i v a l e n t c a t i o n - c o n t a i n i n g b u f f e r i s perhaps p r e f e r a b l e to g e l f i l t r a -t i o n s i n c e the d e t e r g e n t i s removed and t h e c a t i o n added s i m u l t a n e o u s l y . T h i s s i m u l t a n e o u s removal and a d d i t i o n o f d e t e r g e n t and d i v a l e n t c a t i o n r e s p e c t i v e l y seems to f a v o u r v e s i c l e f o r m a t i o n . Magnesium appears to g i v e the b e s t r e -s u l t s i n r e c o n s t i t u t i o n , w i t h the r e a g g r e g a t e formed i n i t s p r e s e n c e b e i n g c l o s e s t i n appearance to the o r i g i n a l membrane. C a l c i u m and t r i v a l e n t c a t i o n s can a l s o f a v o u r the recombina-t i o n o f s o l u b i l i z e d membrane components, b u t the r e s u l t i n g r e a g g r e g a t e s are f r e q u e n t l y amorphous r a t h e r than membran-ous. I t i s a l s o found t h a t the l i p i d - t o - p r o t e i n r a t i o i n r e c o n s t i t u t e d m i c r o b i a l membranes i s dependent on the con-c e n t r a t i o n o f d i v a l e n t c a t i o n used i n r e a g g r e g a t i o n . 3 I n a d d i t i o n t o m a i n t a i n i n g membrane s t r u c t u r e , d i v a l e n t c a t i o n s a r e a l s o i m p o r t a n t i n r e g u l a t i n g membrane p e r m e a b i -2 + l i t y . D e c r e a s e d Ca l e v e l s h a v e b e e n f o u n d t o i n c r e a s e t h e r a t e o f s w e l l i n g o f s e a u r c h i n eggs i n h y p o t o n i c m e d i a , and a l s o t o i n c r e a s e t h e p e r m e a b i l i t y o f m u s c l e and k i d n e y t u b u l e c e l l s t o w a t e r (4). The i n c r e a s e i n N a + p e r m e a b i l i t y o f e r y t h r o c y t e s r e s u l t i n g f r o m t r e a t m e n t w i t h b u t a n o l c a n be 2+ 2+ p a r t i a l l y r e v e r s e d by t h e d i v a l e n t c a t i o n s Ca ,Sr , o r 2+ 2+ Ba , a l t h o u g h Mg has no e f f e c t ( 5 ) . The i n f l u e n c e o f d i v a l e n t c a t i o n s on membrane p e r m e a b i l i t y may be r e l e v a n t t o t h e e l e c t r i c a l p r o p e r t i e s o f e x c i t a b l e c e l l s , s i n c e i n i -t i a t i o n o f an i m p u l s e i s b e l i e v e d t o de p e n d on a change i n t h e membrane p e r m e a b i l i t y t o N a + ( 6 ) . I n f a c t , one t h e o r y o f t h e m o l e c u l a r mechanisms g o v e r n i n g t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l s a s c r i b e s a r o l e t o d i v a l e n t c a t i o n - i n d u c e d c h a n g e s i n membrane s t r u c t u r e a n d / o r h y d r a t i o n , w i t h r e -s u l t a n t c h a n g e s i n membrane i o n p e r m e a b i l i t y c h a r a c t e r i s t i c s ( 7 ) . D i v a l e n t c a t i o n s have a l s o b e en i m p l i c a t e d i n membrane f u s i o n . The p r o c e s s o f membrane f u s i o n may be c o n s i d e r e d t o c o n s i s t o f f o u r s t e p s (8): a) c o n t a c t , i n f l u e n c e d p r i m -a r i l y by t h e e n e r g y b a r r i e r o p p o s i n g d i r e c t a p p o s i t i o n o f t h e membranes; b) i n d u c t i o n , i n v o l v i n g t h e d i s p l a c e m e n t o f 2 + membrane-bound Ca , t h u s i n c r e a s i n g t h e m o t i o n a l f r e e d o m and s t r u c t u r a l t r a n s f o r m a t i o n o f membrane l i p i d s ; c ) f u s i o n , i n v o l v i n g t h e e s t a b l i s h m e n t o f s t a b l e i n t e r m e m b r a n e l i n k a g e s ; and d) s t a b i l i z a t i o n , t h e r e c o v e r y o f t h e newly f u s e d mem-brane to normal conditions by reversal of step b). Model studies on phospholipid vesicles have demonstrated that 2 + cations such as Ca can cause such v e s i c l e s to aggregate (9) . Divalent cations have also been found to increase aggregation of normal red blood c e l l s i n dextrans, probably by decreasing the surface potential of the red blood c e l l s (10) . Thus divalent cations are e f f e c t i v e i n i n i t i a t i n g the process of membrane fusion. Furthermore, spin l a b e l l i n g studies of phospholipid v e s i c l e s (11) have indicated that intermixing of the phospholipids occurs when the ves i c u l a r membranes come into contact by c o l l i s i o n , and that the pre-sence of divalent cations tends to increase the transfer rate of phospholipids between v e s i c l e s . This i s thought to be the r e s u l t of an increased time of membrane contact i n the presence of divalent cations, caused by either of two mech-anisms: a) hydrophobic bonding between ve s i c l e s as a r e s u l t of divalent cation-induced formation of phospholipid aggre-gates within the ve s i c l e s , the surface of which aggregates are repulsive to water (12), or b) divalent cation bridging between v e s i c l e s . Fusion i s believed to occur by the same mechanism as phospholipid transfer, except that i t would involve the movement of a larger number of molecules occur-r i n g i n a larger membrane area. The e f f e c t s of divalent cations on fusion are of considerable importance since mem-brane fusion may well be involved i n the release of a vari e t y of enzymes, hormones, and neurotransmitters (8,13). The marked e f f e c t of divalent cations on the neural 5 r e l e a s e o f t r a n s m i t t e r s has p r o v i d e d one means o f a n a l y z i n g the s t r u c t u r a l and f u n c t i o n a l consequences o f membrane-cation 2+ 2+ i n t e r a c t i o n . I n t r a c e l l u l a r a p p l i c a t i o n o f Ca o r Sr i n the s q u i d g i a n t axon was found to cause a r e l e a s e o f the t r a n s m i t t e r a c e t y l c h o l i n e ( 1 4 ) . T h i s r e l e a s e was u n a f f e c t e d 2+ 2 + by the r e p l a c e m e n t o f e x t e r n a l Ca by Mn o r by the a d d i -t i o n o f L a ^ + to the e x t e r n a l f l u i d medium, p r o c e d u r e s which n o r m a l l y a b o l i s h t r a n s m i t t e r r e l e a s e evoked by p r e s y n a p t i c membrane d e p o l a r i z a t i o n . I n t r a c e l l u l a r i n j e c t i o n o f M n 2 + 2 + o r Mg , on the o t h e r hand, caused e s s e n t i a l l y no r e l e a s e o f 2 + t r a n s m i t t e r , and, i n c o m b i n a t i o n w i t h i n t r a c e l l u l a r Ca i n -j e c t i o n , caused a s l i g h t d e c r e a s e i n the amount o f t r a n s -2 + m i t t e r r e l e a s e d by Ca . S t u d i e s o f t r a n s m i t t e r r e l e a s e a t the s k e l e t a l muscle neuromuscular j u n c t i o n (15) demonstrate 2 + the i m p o r t a n c e o f Ca i n c o u p l i n g nerve t e r m i n a l d e p o l a r i -z a t i o n w i t h a c e t y l c h o l i n e r e l e a s e . S t r o n t i u m can r e p l a c e 2 + Ca i n t h i s p r o c e s s , b u t i s somewhat l e s s e f f e c t i v e . The 2+ ' 2+ 2+ pr e s e n c e o f Mg i n h i b i t s b o t h the Ca - and Sr - i n d u c e d t r a n s m i t t e r r e l e a s e . I n a d d i t i o n t o t h i s r o l e o f d i v a l e n t c a t i o n s i n d e p o l a r -i z a t i o n - s e c r e t i o n c o u p l i n g , t h e r e i s e v i d e n c e t h a t t h e s e i o n s may a l s o c o n t r i b u t e d i r e c t l y to e l e c t r i c a l changes i n e x c i t a b l e t i s s u e . I n b o t h b a r n a c l e muscle f i b r e s (16) and mammalian m y o c a r d i a l f i b r e s ( 1 7 ) , the d i v a l e n t c a t i o n C a 2 + has been shown t o be a c a r r i e r o f membrane c u r r e n t . C a l c i u m can be s u b s t i t u t e d i n b o t h cases by o t h e r a l k a l i n e e a r t h i o n s , 2+ 2 + such as Ba o r Sr , which w i l l a l s o a c t as membrane c u r r e n t 6 c a r r i e r s . A t a m o l e c u l a r l e v e l , two d i f f e r e n t t h e o r i e s have been s u g g e s t e d t o e x p l a i n the a c t i o n s o f c a t i o n s . One theory-s t a t e s t h a t the changes noted w i t h d i v a l e n t c a t i o n s can be e x p l a i n e d by a s c r e e n i n g p r o c e s s . I n c r a y f i s h axons ( 1 8 ) , the same e x t r a c e l l u l a r c o n c e n t r a t i o n s o f s e v e r a l d i v a l e n t , ,. _ 2+ M 2+ 0 2+ „ 2+ „ 2+ , „.2+ i o n s , i n c l u d i n g Ca , Mg , S r , Ba , Co , and N i , were found t o cause the same degree o f p o s i t i v e s h i f t i n the t h r e s h o l d membrane p o t e n t i a l f o r s p i k e i n i t i a t i o n . T r i -v a l e n t c a t i o n s were more e f f e c t i v e i n p r o d u c i n g t h i s s h i f t t h a n d i v a l e n t c a t i o n s , b u t a g a i n , no d i f f e r e n c e s were n o t e d among the t r i v a l e n t c a t i o n s t e s t e d . T h i s l a c k o f d i f f e r e n -t i a t i o n among i o n s o f t h e same v a l e n c e i s one o f t h e major p r e d i c t i o n s o f a s i m p l e s c r e e n i n g t h e o r y . The o p e r a t i o n o f a s c r e e n i n g mechanism a l s o p r e d i c t s the e x i s t e n c e o f a l i n e a r r e l a t i o n s h i p between t h e t h r e s h o l d p o t e n t i a l s h i f t and the l o g a r i t h m o f the c a t i o n c o n c e n t r a t i o n , w i t h the s l o p e o f t h i s l i n e p r e d i c t e d by the Gouy e q u a t i o n f o r s u r -f a c e p o t e n t i a l . These p r e d i c t i o n s were f o u n d t o be borne ou t i n the c r a y f i s h axon, l e n d i n g f u r t h e r s u p p o r t t o the e x p l a n a t i o n o f d i v a l e n t c a t i o n e f f e c t s i n terms o f a s i m p l e s c r e e n i n g mechanism, a l t h o u g h o t h e r e x p l a n a t i o n s c o u l d n o t be r u l e d o u t . S t u d i e s o f the i n f l u e n c e of d i v a l e n t c a t i o n s on the s u r f a c e p o t e n t i a l o f p h o s p h o l i p i d b i l a y e r s ( 1 9 ) , as deduced from conductance measurements o f a n e u t r a l c a t i o n c a r r i e r , have shown t h a t t h e s e e f f e c t s can be e x p l a i n e d l a r g e l y on 7 the b a s i s o f a s c r e e n i n g mechanism. W i t h p h o s p h a t i d y l -s e r i n e b i l a y e r s , complete agreement between e x p e r i m e n t a l c u r v e s and t h e o r e t i c a l c u r v e s based s o l e l y on s c r e e n i n g was o b t a i n e d w i t h S r 2 + and B a 2 + . F o r C a 2 + and Mg 2 +, however, the p o s s i b i l i t y o f b i n d i n g had t o be i n c l u d e d i n o r d e r t o f i t t he d a t a t o the t h e o r e t i c a l c u r v e s , a l t h o u g h the a s s o c -i a t i o n c o n s t a n t s a re e x t r e m e l y low. With p h o s p h a t i d y l -g l y c e r o l b i l a y e r s , s i m i l a r r e s u l t s were o b t a i n e d , e x c e p t 2 + t h a t i t was n e c e s s a r y t o p o s t u l a t e b i n d i n g o n l y w i t h Ca , and the d e v i a t i o n from the t h e o r e t i c a l s c r e e n i n g c u r v e w i t h C a 2 + was g r e a t e r i n p h o s p h a t i d y l g l y c e r o l b i l a y e r s than i n p h o s p h a t i d y l s e r i n e b i l a y e r s . No changes i n the s u r f a c e po-t e n t i a l o f n e u t r a l b i l a y e r s composed o f 7 - d e h y d r o c h o l e s t e r o l o f p h o s p h a t i d y l e t h a n o l a m i n e were produced by d i v a l e n t c a t i o n s . These c o n s i d e r a t i o n s have a b e a r i n g on t h e changes i n 1-a n i l i n o n a p h t h a l e n e - 8 - s u l f o n a t e (ANS) f l u o r e s c e n c e produced i n such l i p i d systems when d i v a l e n t c a t i o n s a r e p r e s e n t . The enhancement o f ANS f l u o r e s c e n c e by d i v a l e n t c a t i o n s , which has u s u a l l y been e x p l a i n e d i n terms o f charge n e u t r a l i z a t i o n o f p h o s p h o l i p i d a n i o n i c groups by the i o n s , may i n v o l v e a s c r e e n i n g component whereby t h e a d d i t i o n o f d i v a l e n t i o n s would s e r v e to d e c r e a s e the n e g a t i v e s u r f a c e p o t e n t i a l gen-e r a t e d by the a d s o r p t i o n o f n e g a t i v e l y charged ANS m o l e c u l e s and t e n d i n g t o d e c r e a s e f u r t h e r ANS b i n d i n g as a r e s u l t o f e l e c t r o s t a t i c r e p u l s i v e f o r c e s . The a d d i t i o n o f d i v a l e n t c a t i o n s would, t h e r e f o r e , s c r e e n the s u r f a c e charge and a l l o w more ANS to be adsorbed. 8 An a l t e r n a t e t h e o r y of membrane-cation i n t e r a c t i o n s t a t e s t h a t d i v a l e n t c a t i o n s b i n d i n some manner to some component(s) o f the membrane and t h i s i s r e s p o n s i b l e f o r the e f f e c t s noted. C a l c i u m i o n s a r e known to b i n d to human e r y t h r o c y t e membranes ( 2 0 ) , i n a manner s u g g e s t i n g t h a t the b i n d i n g s i t e s are n o n - s p e c i f i c and i o n i c i n n a t u r e . I t i s b e l i e v e d t h a t t h e s e b i n d i n g s i t e s c o u l d be the phosphate groups o f p h o s p h o l i p i d s o r p o l y p h o s p h o i n o s i t i d e s and s i a l i c a c i d c a r b o x y l groups on the membrane s u r f a c e . F u r t h e r i n -v e s t i g a t i o n s o f t h i s t h e o r y (21,22,23) have s e r v e d to con-f i r m t h a t d i v a l e n t Ions can b i n d to p h o s p h o l i p i d s , and the i n v e s t i g a t o r s have proposed models f o r the c a t i o n - l i p i d com-p l e x , i n v o l v i n g the i n t e r a c t i o n o f one d i v a l e n t c a t i o n w i t h the phosphate groups of e i t h e r two o f f o u r p h o s p h o l i p i d m o l e c u l e s . I t has a l s o been proposed t h a t d i v a l e n t c a t i o n s may form a p r o t e i n - p h o s p h o l i p i d complex by b r i d g i n g n e g a t i v e s i t e s o f l i p i d and p r o t e i n . T h i s type o f complex was shown to be formed i n a model system c o n s i s t i n g o f p h o s p h a t i d y l -2+ 2 + s e r i n e and g l y c y l g l y c i n e i n the p r e s e n c e o f Ca , Mg , M n 2 + , and S r 2 + , a l t h o u g h n o t w i t h B a 2 + , C u 2 + , N l 2 + , o r Z n 2 + 2 + (24) . I n a n o t h e r system, Ca and s p e c t r i n were found to i n t e r a c t s y n e r g i s t i c a l l y i n c a u s i n g an i n c r e a s e i n perme-a b i l i t y o f p h o s p h a t i d y l s e r i n e v e s i c l e s t o N a + (25). Two 2 + models were proposed to e x p l a i n t h i s e f f e c t : Ca b i n d i n g to a n i o n i c s i t e s on b o t h the p r o t e i n and t h e v e s i c l e s would f a c i l i t a t e p r o t e i n p e n e t r a t i o n e i t h e r by d e c r e a s i n g e l e c t r o -9 s t a t i c r e p u l s i o n s , o r by c a u s i n g a c o n f o r m a t i o n a l change i n the p r o t e i n which f a v o u r e d p e n e t r a t i o n . A l t e r n a t i v e l y , the 2 + f o r m a t i o n o f t r i d e n t a t e complexes i n v o l v i n g p r o t e i n , Ca , and the n e g a t i v e l y charged v e s i c l e s c o u l d i n d u c e l o c a l p e r -t u r b a t i o n s l e a d i n g t o d r a s t i c changes i n p e r m e a b i l i t y , A number o f t e c h n i g u e s have been employed to t r y to r e s o l v e the q u e s t i o n s o f how d i v a l e n t c a t i o n s i n t e r a c t w i t h membranes, what changes th e s e i n t e r a c t i o n s i n d u c e i n the membrane, and how these changes r e l a t e to p h y s i o l o g i c a l p r o c e s s e s . Many s t u d i e s have been conducted on s i m p l e model p h o s p h o l i p i d systems, c o n s i s t i n g f r e q u e n t l y of a - s i n g l e w e l l - d e f i n e d l i p i d m o l e c u l e . The r e l e v a n c e o f such s t u d i e s to b i o l o g i c a l membranes, which e x h i b i t a complex h e t e r o -g e n e i t y i n p h o s p h o l i p i d c o m p o s i t i o n ' s q u e s t i o n a b l e . F u r t h e r , such s t u d i e s p r e c l u d e an a n a l y s i s o f t h e r o l e o f membrane p r o t e i n s i n m e d i a t i n g the s t r u c t u r a l and f u n c t i o n a l conse-quences o f membrane-cation i n t e r a c t i o n . A l t e r n a t i v e l y , the membrane e f f e c t s o f i n o r g a n i c c a t i o n s may be a n a l y z e d i n terms o f ion-dependent membrane-associated enzymes, such as t r a n s p o r t ATPase. The k i n e t i c c o m p l e x i t i e s a r i s i n g from the p a r t i c u l a t e n a t u r e of t h e s e enzymes, however, a r e such t h a t d e t a i l e d i n f o r m a t i o n on the r o l e o f c a t i o n s i n m o d u l a t i n g t h e s e p r o c e s s e s i s l a r g e l y i n d i r e c t and u n c e r t a i n . S p i n l a b e l l i n g s t u d i e s have y i e l d e d i n f o r m a t i o n on the e f f e c t s o f d i v a l e n t c a t i o n s on membranes. I t has been demon-s t r a t e d i n b a c t e r i a l c y t o p l a s m i c membranes (26) t h a t c a t i o n s i n c r e a s e membrane r i g i d i t y by two mechanisms. One mechanism o c c u r s a t low i o n i c c o n c e n t r a t i o n and i s e x p l a i n e d i n terms of c a t i o n b i n d i n g to p h o s p h o l i p i d head groups, and would i n -v o l v e n e u t r a l i z a t i o n o f n e t n e g a t i v e s u r f a c e charge and e l i m i n a t i o n o f r e p u l s i v e f o r c e s between l i p i d head groups so t h a t the membrane becomes more r i g i d . A t h i g h i o n i c con-c e n t r a t i o n , a r e v e r s i b l e e f f e c t comes i n t o o p e r a t i o n whereby f u r t h e r r i g i d i t y can be i n d u c e d by c a t i o n s . T h i s i s ex-p l a i n e d i n terms o f i o n i c e f f e c t s on water s t r u c t u r e b e i n g t r a n s m i t t e d t o the membrane s u r f a c e . NMR s t u d i e s (27) have a l s o i m p l i c a t e d changes i n water s t r u c t u r e i n the membrane as a r e s u l t o f d i v a l e n t c a t i o n i n t e r a c t i o n . U s i n g two s p i n - l a b e l analogues ( 2 8 ) , one o f which probes the p o l a r r e g i o n o f the l i p i d b i l a y e r and the o t h e r a more h y d r o p h o b i c r e g i o n , i t has been p o s s i b l e to demon-s t r a t e t h a t C a 2 + d e c r e a s e s the m o t i o n a l freedom o f f a t t y a c i d c h a i n s near the p o l a r head groups i n b o t h p h o s p h a t i d y l -s e r i n e and p h o s p h a t i d y l i n o s i t o l membranes. C a l c i u m a l s o de-c r e a s e s the f l u i d i t y o f the r e g i o n near the a p o l a r end of the f a t t y a c i d c h a i n s i n p h o s p h a t i d y l s e r i n e , b u t has no d e t e c t a b l e e f f e c t i n the h y d r o p h o b i c r e g i o n s o f p h o s p h a t i d y l i n o s i t o l membranes, p o s s i b l y because o f the c a r b o n - c a r b o n double bond i n the f a t t y a c i d c h a i n s o f p h o s p h a t i d y l i n o s i t o l w hich may p r e v e n t t i g h t p a c k i n g o f the l i p i d c h a i n s i n the a p o l a r r e g i o n o f the membrane, o r may d e c r e a s e the a f f i n i t y o f the l i p i d f o r C a 2 + . I t has a l s o been found t h a t d i v a l e n t i o n s such as 2+ 2+ 2+ Ca , Ba , and S r tend t o cause phase s e p a r a t i o n s o f 11 c e r t a i n p h o s p h o l i p i d s i n p h o s p h o l i p i d membranes, such as l e c i t h i n i n p h o s p h a t i d y l s e r i n e b i l a y e r s , p h o s p h a t i d i c a c i d i n p h o s p h a t i d y l c h o l i n e membranes and p h o s p h a t i d y l c h o l i n e i n p h o s p h a t i d i c a c i d membranes (29,30). T h i s c l u s t e r i n g e f f e c t shows some degree o f i o n i c s e l e c t i v i t y , and i t has been s u g g e s t e d t h a t t h e s e f i n d i n g s may be s i g n i f i c a n t i n some p h y s i o l o g i c a l e v e nts such as p e r m e a b i l i t y changes o r e x c i t a -t i o n . P h o s p h o l i p i d b i l a y e r s a r e known to undergo a change from a r e l a t i v e l y o r d e r e d s t r u c t u r e t o a more f l u i d s t a t e a t a c h a r a c t e r i s t i c t r a n s i t i o n t e mperature. T h i s t r a n s i t i o n t e m perature can be markedly a f f e c t e d by s e v e r a l f a c t o r s i n -c l u d i n g pH and the i o n i c c o m p o s i t i o n o f the s u r r o u n d i n g 2+ 2+ medium. Both Mg and Ca have been found t o i n c r e a s e the t r a n s i t i o n t e m p e r a t u r e o f n e g a t i v e l y c h a r g e d p h o s p h o l i p i d b i l a y e r s , i n d i c a t i n g an i n c r e a s e i n membrane s t a b i l i t y (31, 32 ) , p r o b a b l y by charge n e u t r a l i z a t i o n . A more d e t a i l e d 2+ s t u d y on the e f f e c t o f Ca on the t r a n s i t i o n t e mperature o f p h o s p h a t i d y l g l y c e r o l (33) has shown t h a t a low c o n c e n t r a -2 + t i o n o f Ca markedly i n c r e a s e s the t r a n s i t i o n t e m p e r a t u r e , 2 + bu t on i n c r e a s i n g the Ca c o n c e n t r a t i o n even h i g h e r , t h e r e 2 + i s l i t t l e f u r t h e r e f f e c t u n t i l the Ca : p h o s p h a t i d y l g l y c e r o l r a t i o exceeds 1:2, whereupon an a d d i t i o n a l l a r g e i n c r e a s e i n the t r a n s i t i o n t emperature o c c u r s . There i s a n o t i c e a b l e v i s i b l e d i f f e r e n c e when t h i s r a t i o i s exceeded; p r e c i p i t a -t i o n can be seen to o c c u r i n the m i l k y p h o s p h o l i p i d d i s p e r -s i o n . These s t r u c t u r a l a l t e r a t i o n s may be v i s u a l i z e d i n more d e t a i l by t h e f r e e z e - e t c h i n g t e c h n i q u e . When t h e Ca : p h o s -p h a t i d y l g l y c e r o l r a t i o i s l e s s t h a n 1:2, l i p o s o m a l s t r u c -t u r e s w i t h s m o o t h f r a c t u r e f a c e s a r e s e e n . A t h i g h e r r a t i o s , c y l i n d r i c a l l a m e l l a r s t r u c t u r e s a p p e a r . These f i n d i n g s a r e 2 + t h o u g h t t o i n d i c a t e t h a t a t Ca : p h o s p h a t i d y l g l y c e r o l r a t i o s 2 + o f l e s s t h a n 1:2, Ca n e u t r a l i z a t i o n o f t h e p h o s p h o l i p i d c h a r g e p r o d u c e s an i n c r e a s e i n m o l e c u l a r p a c k i n g , r e s u l t i n g i n an i n c r e a s e d t r a n s i t i o n t e m p e r a t u r e . When t h e r a t i o i s 2 + g r e a t e r t h a n 1:2, a s t a b l e c o m p l e x i s f o r m e d b e t w e e n Ca and two p h o s p h a t i d y l g l y c e r o l m o l e c u l e s , t h u s i n d u c i n g e v e n g r e a t e r o r d e r i n t h e b i l a y e r . The compound l - a n i l i n o n a p h t h a l e n e - 8 - s u l f o n a t e ( A N S ) , w h i c h w i l l e x h i b i t e n h a n c e d f l u o r e s c e n c e i n n o n - p o l a r e n -v i r o n m e n t s , h a s b e e n u s e d as a p r o b e t o d e t e r m i n e t h e p e r -t u r b a t i o n a l e f f e c t s o f c a t i o n s o n membranes. ANS e x h i b i t s an e n h a n c e d f l u o r e s c e n c e s i g n a l i n t h e p r e s e n c e o f e r y t h r o -c y t e membranes, s k e l e t a l m u s c l e m i c r o s o m e s , a n d d i s p e r s i o n s o f p h o s p h a t i d y l c h o l i n e , l y s o p h o s p h a t i d y l c h o l i n e , p h o s p h a t i d y l e t h a n o l a m i n e , a n d p h o s p h a t i d y l s e r i n e as c o m p a r e d w i t h a p u r e l y a q u e o u s medium (34,35). When i n o r g a n i c c a t i o n s a r e a d d e d , an i n c r e a s e i n ANS f l u o r e s c e n c e i n t e n s i t y i s n o t e d . T h i s i n c r e a s e i s g r e a t e s t w i t h t r i v a l e n t c a t i o n s , i n t e r -m e d i a t e w i t h d i v a l e n t c a t i o n s , a n d l e a s t w i t h m o n o v a l e n t c a t i o n s . No d i f f e r e n c e s w e re n o t e d among c a t i o n s o f t h e same v a l e n c e . T h i s e n h a n c e m e n t o f f l u o r e s c e n c e may b e due e i t h e r t o a n i n c r e a s e i n t h e h y d r o p h o b i c c h a r a c t e r o f t h e membrane i n t h e p r e s e n c e o f c a t i o n s o r t o an i n c r e a s e i n ANS b i n d i n g due t o a n e u t r a l i z a t i o n o f n e g a t i v e c h a r g e s on t h e membrane s u r f a c e by t h e c a t i o n s . A l a t e r s t u d y o f ANS f l u o r e s c e n c e i n l i p i d v e s i c l e s (36) gave s i m i l a r r e s u l t s , e x c e p t t h a t d i f f e r e n c e s were n o t e d among t h e v a r i o u s d i v a l e n t c a t i o n s t e s t e d . G e n e r a l l y , t h e f l u o r e s c e n c e i n c r e a s e d w i t h d e c r e a s i n g i o n i c r a d i u s , e x c e p t f o r M g 2 + w h i c h c a u s e d an anomalous d e c r e a s e . The e f f e c t i v e n e s s o f d i v a l e n t c a t i o n s i n i n c r e a s i n g ANS f l u o r e s c e n c e was f o u n d t o p a r a l l e l t h e s t a b i -l i t y o f c h e l a t e c o m p l e x e s f o r m e d by t h e s e c a t i o n s , e v e n t o 2 + t h e anomalous d e c r e a s e w i t h Mg , s u g g e s t i n g t h a t t h e c a t i o n s may c h e l a t e t o t h e l i p i d p h o s p h a t e g r o u p s , t h u s f a c i l i t a t i n g ANS b i n d i n g . Most o f t h e above s t u d i e s o f d i v a l e n t c a t i o n a c t i o n a t t h e m o l e c u l a r l e v e l , however, have shown l i t t l e o r no i o n i c s e l e c t i v i t y , u n l i k e t h e m a j o r i t y o f c a t i o n - d e p e n d e n t p h y s i o -l o g i c a l p r o c e s s e s w h i c h t e n d t o be r e l a t i v e l y i o n s p e c i f i c . S i n c e most o f t h e s e s t u d i e s were c o n d u c t e d on model p h o s -p h o l i p i d s y s t e m s , i t i s p o s s i b l e t h a t t h e l a c k o f s e l e c t i -v i t y may be r e l a t e d t o t h e a b s e n c e o f membrane p r o t e i n s . The u s e o f r e a l b i o l o g i c a l membranes c o u l d p e r h a p s r e s o l v e some o f t h e d i f f i c u l t i e s i n r e l a t i n g t h e m o l e c u l a r a c t i o n o f c a t i o n s t o v a r i o u s p h y s i o l o g i c a l p r o c e s s e s , b u t t h e p r o b l e m t h e n a r i s e s t h a t w i t h t h e above m e n t i o n e d t e c h n i q u e s , i t i s d i f f i c u l t , i f n o t i m p o s s i b l e , t o d i s t i n g u i s h t h e r e l a t i v e c o n t r i b u t i o n s o f t h e p r o t e i n and l i p i d components t o c a t i o n i n t e r a c t i o n s c a u s i n g a l t e r a t i o n s i n t h e membrane s t r u c t u r e . P r e v i o u s work (37,38) h a s , d e m o n s t r a t e d t h e u s e f u l n e s s o f t h e p r o b e t r i n i t r o b e n z e n e s u l f o n i c a c i d (TNBS), a compound w h i c h r e a c t s s p e c i f i c a l l y w i t h p r i m a r y amino g r o u p s f o r m i n g a c h r o m o p h o r i c t r i n i t r o p h e n y l a t e d d e r i v a t i v e , i n i n v e s t i -g a t i n g membrane p e r t u r b a t i o n s i n d u c e d by d r u g s o r c a t i o n s . T h i s compound has a d i s t i n c t a d v a n t a g e o v e r o t n e r c h e m i c a l membrane p r o b e s , s u c h as ANS, b e c a u s e o f t h e s p e c i f i c i t y o f i t s r e a c t i o n and t h e s t a b i l i t y o f t h e d e r i v a t i v e f o r m e d . T h i s makes i t p o s s i b l e t o d e t e r m i n e e x a c t l y w h i c h components o f t h e membrane a r e b e i n g a f f e c t e d by d r u g - o r c a t i o n - i n d u c e d p e r t u r b a t i o n s . I n t h e f o l l o w i n g s t u d y , TNBS and o t h e r c h e m i c a l p r o b e s have been u s e d t o c h a r a c t e r i z e c a t i o n - i n d u c e d membrane p e r t u r b a t i o n s , i n terms o f t h e a c c e s s i b i l i t y a n d / o r r e a c t i v i t y o f membrane amino g r o u p s , and i n terms o f t h e r e l a t i v e i n c o r p o r a t i o n o f p r o b e s i n t o t h e membrane p r o t e i n s and p h o s p h o l i p i d s . A t t e m p t s were a l s o made t o c o r r e l a t e t h e s t r u c t u r a l c h a n g e s w i t h f u n c t i o n a l c h a n g e s i n t h e membrane a s , f o r example, by s t u d y i n g t h e e f f e c t s o f t h e c a t i o n s on t h e s u s c e p t i b i l i t y o f e r y t h r o c y t e s t o h y p o t o n i c a l l y - i n d u c e d h e m o l y s i s . I t was hoped t h a t t h e s e model s t u d i e s w i t h e r y t h r o c y t e s w o u l d p r o v i d e a g r e a t e r u n d e r s t a n d i n g o f m o l e -c u l a r a s p e c t s o f i n o r g a n i c c a t i o n i n t e r a c t i o n w i t h membranes i n g e n e r a l . MATERIALS The f o l l o w i n g c h e m i c a l s and enzymes were o b t a i n e d f r o m t h e Sigma C h e m i c a l Company: t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ( T r i z m a b a s e ) , n i t r o p h e n y l p h o s p h a t e , i m i d a z o l e (Grade I I I ) , 5 , 5 ' - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB), a c e t y l t h i o -c h o l i n e c h l o r i d e , s t a n d a r d p h o s p h o r u s s o l u t i o n ( 2 0 y * g / m l ) , c h o l e s t e r o l , N - a c e t y l n e u r a m i n i c a c i d (Type IV) u s e d as a s t a n d a r d i n t h e s i a l i c a c i d a s s a y , t r y p s i n (2x c r y s t a l l i z e d , Type I I I , f r o m b o v i n e p a n c r e a s ) , P h o s p h o l i p a s e A ( N a j a n a j a venom), and p i c r y l s u l f o n i c a c i d (TNBS). B o v i n e a l b u m i n ( F r a c t i o n V f r o m b o v i n e p l a s m a ) , u s e d as t h e s t a n d a r d i n t h e p r o t e i n a s s a y , was o b t a i n e d f r o m Armour P h a r m a c e u t i c a l s . U l t r a p u r e enzyme g r a d e s u c r o s e was p u r c h a s e d f r o m Schwarz/Mann. N e u r a m i n i d a s e ( V i b r i o comma ( c h o l e r a e ) ) came f r o m B e h r i n g w e r k e . Sephadex G-200" was p u r -c h a s e d f r o m P h a r m a c i a and p i c r y l c h l o r i d e f r o m M a t h e s o n Coleman and B e l l . A l l o t h e r c h e m i c a l s u s e d were o f r e a g e n t g r a d e q u a l i t y . METHODS MEMBRANE PREPARATIONS a) E r y t h r o c y t e Membranes E r y t h r o c y t e membranes were p r e p a r e d f r o m o u t d a t e d human b l o o d s t o r e d i n a c i d - c i t r a t e - d e x t r o s e by a s t e p - w i s e h e m o l y s i s p r o c e d u r e ( 3 9 ) . A u n i t o f b l o o d ( a p p r o x i m a t e l y 450 ml) was d i l u t e d t o 1200 ml w i t h i s o t o n i c s a l i n e and c e n t r i f u g e d a t 650 x g f o r 5 m i n u t e s to remove t h e p l a s m a and b u f f y c o a t . T h i s w a s h i n g was r e p e a t e d once. A p p r o x i -m a t e l y 100 ml o f p a c k e d r e d b l o o d c e l l s were t h e n p r o c e s s e d f u r t h e r by d i l u t i o n t o 1200 ml w i t h 0.08 M sodium c h l o r i d e . T h i s m i x t u r e was s t i r r e d a t 4°C f o r 10 m i n u t e s , t h e n c e n t r i -f u g e d a t 16,000 x g f o r 5 m i n u t e s . The s u p e r n a t a n t was removed and t h e p r o c e d u r e r e p e a t e d w i t h 0.06 M, 0.04 M, 0.02 M, and 0.009 M sodium c h l o r i d e . A t t h e l a s t two s o d i u m c h l o r i d e c o n c e n t r a t i o n s , t h e pH was a d j u s t e d t o 7.4 w i t h 0.5 M t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ( T r i s ) b e f o r e s t i r r i n g . The f i n a l s t e p i n v o l v e d s t i r r i n g w i t h 10 mM T r i s pH 7.4, t h e n c e n t r i f u g a t i o n a t 40,000 x g f o r 10 m i n u t e s . The e r y t h r o -c y t e s were d i l u t e d t o a f i n a l p r o t e i n c o n c e n t r a t i o n o f 3 -5 mg/ml, q u i c k f r o z e n u s i n g d r y i c e / a c e t o n e , and s t o r e d a t -20°C. b) B r a i n S y n a p t i c V e s i c l e s R a t b r a i n s y n a p t i c v e s i c l e s were p r e p a r e d by t h e method o f C o r b e t t e t a l ( 4 0 ) , w i t h some m i n o r m o d i f i c a t i o n s . Two a d u l t m a l e W i s t a r r a t s ( 250-300 g r a m s ) were k i l l e d b y d e -c a p i t a t i o n a n d t h e b r a i n s r e moved. The w h o l e b r a i n s ( a p p r o x i -m a t e l y 3 grams w e t w e i g h t ) w e re h o m o g e n i z e d i n 30 ml c o l d 0.32 M s u c r o s e a nd c e n t r i f u g e d f o r 10 m i n u t e s a t 1000 x g. The s u p e r n a t a n t was r e t a i n e d a nd t h e p e l l e t r e s u s p e n d e d b y h o m o g e n i z a t i o n i n 15 ml c o l d 0.32 M s u c r o s e a n d r e c e n t r i f u g e d a t 1000 x g. The two s u p e r n a t a n t s w e re c o m b i n e d and c e n t r i -f u g e d a t 10,000 x g f o r 20 m i n u t e s . The p e l l e t o b t a i n e d was r e s u s p e n d e d i n 5 ml c o l d 0.32 M s u c r o s e , t h e n 25 ml 5 mM T r i s pH 7.4, c o n t a i n i n g 1 mM e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) were a d d e d a n d t h e m i x t u r e s t i r r e d i n i c e f o r 45 m i n u t e s . T h i s was f o l l o w e d b y c e n t r i f u g i n g a t 11,500 x g f o r 20 m i n u t e s . The s u p e r n a t a n t was f i l t e r e d t h r o u g h 355 nm, 300 "nm, and 200 nm f i l t e r s i n s u c c e s s i o n , t h e n c e n t r i f u g e d a t 100,000 x g f o r 30 m i n u t e s . The f i n a l p e l l e t was r e s u s -p e n d e d i n 5 mM T r i s pH 7.4, q u i c k f r o z e n u s i n g d r y i c e / a c e t o n e , a nd s t o r e d a t -20°C. B o v i n e s y n a p t i c v e s i c l e s w e r e p r e p a r e d b y t h e same me t h o d e x c e p t t h a t o n l y c o r t e x was u s e d . B e e f b r a i n was o b t a i n e d f r e s h f r o m I n t e r c o n t i n e n t a l P a c k e r s L t d . o n t h e day o f t h e p r e p a r a t i o n . c ) R a t L i v e r M i c r o s o m e s R a t l i v e r m i c r o s o m e s w e r e p r e p a r e d b y t h e c a l c i u m p r e -c i p i t a t i o n m e t h o d o f Kamath and N a r a y a n ( 4 1 ) . The a n i m a l s w e r e s t a r v e d f o r 24 h o u r s p r i o r t o t h e s t a r t o f t h e p r e p a r a -t i o n , a n d k i l l e d b y d e c a p i t a t i o n . The l i v e r s w e r e p e r f u s e d w i t h 0.25 M s u c r o s e b u f f e r e d w i t h 10 mM T r i s t o pH 7.5, removed and p l a c e d i n s u c r o s e . The t i s s u e was h o m o g e n i z e d i n t h e b u f f e r e d s u c r o s e s o l u t i o n a t a c o n c e n t r a t i o n o f 25% ( t i s s u e w e i g h t / v o l u m e o f s u c r o s e ) t h e n d i l u t e d t o a 10% s o l u t i o n . The homogenate was c e n t r i f u g e d a t 600 x g f o r 5 m i n u t e s , t h e s u p e r n a t a n t f i l t e r e d t h r o u g h c h e e s e c l o t h , c e n t r i f u g e d f o r 10 m i n u t e s a t 12,000 x g and f i l t e r e d a g a i n t h r o u g h c h e e s e c l o t h . A s u f f i c i e n t volume o f 1 M c a l c i u m c h l o r i d e was added t o t h e s u p e r n a t a n t t o g i v e a f i n a l c a l c i u m c o n c e n t r a t i o n o f 8 mM, and t h e s o l u t i o n was a l l o w e d , t o s t a n d f o r 5 m i n u t e s a t 4°C b e f o r e c e n t r i f u g a t i o n a t 27,000 x g f o r 15 m i n u t e s . The p e l l e t was washed t w i c e by c e n t r i f u g a t i o n w i t h 0.15 M p o t a s s i u m c h l o r i d e and r e s u s p e n d e d i n 0.15 M KC1 t o o n e - h a l f t h e volume o f t h e o r i g i n a l homogenate. The m i c r o s o m e s were q u i c k f r o z e n u s i n g d r y i c e / a c e t o n e and s t o r e d a t -20°C. d) P u r i f i e d R a t L i v e r M i c r o s o m a l Membranes F o r some e x p e r i m e n t s , p u r i f i e d m i c r o s o m a l membranes were p r e p a r e d a c c o r d i n g t o t h e method o f W e i h i n g e t a l ( 4 2 ) . T h i s p r o c e d u r e i n v o l v e d a m u l t i p l e s a l t e x t r a c t i o n o f c r u d e m i c r o -somes t o remove r i b o s o m e s and p r o t e i n a d s o r b e d t o t h e s u r -f a c e o f t h e m i c r o s o m a l v e s i c l e s w h i l e r e t a i n i n g t h e i n t e g r i t y o f t h e m i c r o s o m a l membrane. COMPOSITIONAL ASSAYS P r o t e i n was d e t e r m i n e d by t h e method o f Lowry e t a l (43) w i t h b o v i n e serum a l b u m i n (1 mg/ml) u s e d as t h e s t a n d a r d . P h o s p h o l i p i d was d e t e r m i n e d by B a r t l e t t 1 s m o d i f i c a t i o n o f t h e F i s k e - S u b b a r o w p h o s p h a t e a n a l y s i s ( 4 4 ) . C h o l e s t e r o l was measured by t h e method o f Zak e t a l ( 4 5 ) . S i a l i c a c i d was d e t e r m i n e d by h y d r o l y z i n g t h e sample w i t h 0.1 N H^SO^ a t 8 0 ° C f o r 30 m i n u t e s t h e n a s s a y i n g a c -c o r d i n g t o t h e method o f Warren ( 4 6 ) . ENZYME TREATMENTS a) N e u r a m i n i d a s e E r y t h r o c y t e membranes were t r e a t e d w i t h n e u r a m i n i d a s e ( V i b r i o comma ( c h o l e r a e ) , B e h r i n g w e r k e ) t o remove s i a l i c a c i d i n t h e f o l l o w i n g manner. F o r e a c h ml o f membranes t o be t r e a t e d , 3.0 ml o f 50 mM s o d i u m a c e t a t e b u f f e r pH 5.5, 0.5 ml 20 mM c a l c i u m c h l o r i d e and 20 y x l n e u r a m i n i d a s e (500 u n i t s / m l ) were u s e d . The r e a c t i o n m i x t u r e was i n c u b a t e d a t 37°C f o r 30 m i n u t e s t h e n c e n t r i f u g e d f o r 5 m i n u t e s a t 40,000 x g. The membranes were washed o n c e w i t h c o l d d o u b l e d i s t i l l e d w a t e r and r e s u s p e n d e d i n c o l d d o u b l e d i s t i l l e d w a t e r t o a p p r o x i m a t e l y t h e o r i g i n a l volume. C o n t r o l mem-b r a n e s were t r e a t e d s i m i l a r l y e x c e p t t h a t w a t e r was s u b s t i -t u t e d f o r t h e n e u r a m i n i d a s e . T h i s p r o c e d u r e g e n e r a l l y r e -moved 70% o f t h e t o t a l s i a l i c a c i d . b) T r y p s i n E r y t h r o c y t e membrane p r o t e i n s were a l t e r e d b y t r e a t i n g t h e membranes w i t h t r y p s i n i n t h e f o l l o w i n g manner. Mem-b r a n e s were i n c u b a t e d w i t h t r y p s i n (500y/.g/ml; 0.07 ml t r y p s i n / m l membranes) a t 37°C f o r 15 m i n u t e s , t h e n c e n t r i -f u g e d a t 40,000 x g f o r 5 m i n u t e s . The membranes were washed o n c e w i t h c o l d d o u b l e d i s t i l l e d w a t e r and r e s u s p e n d e d i n c o l d d o u b l e d i s t i l l e d w a t e r t o a p p r o x i m a t e l y o n e - h a l f t h e o r i g i n a l volume. c ) P h o s p h o l i p a s e A E r y t h r o c y t e membranes were t r e a t e d w i t h p h o s p h o l i p a s e A ( N a j a n a j a venom, Sigma) by t h e f o l l o w i n g method. The p h o s p h o l i p a s e A (PLA) s o l u t i o n (10 mg/ml) was f i r s t h e a t e d a t 70°C f o r 10 m i n u t e s t o d e s t r o y p r o t e o l y t i c a c t i v i t y . Then, f o r e a c h 2.0 ml a l i q u o t o f membranes t o be t r e a t e d , 4.0 ml 20 mM T r i s pH 8.0, 1.0 ml 20 mM c a l c i u m c h l o r i d e , and 0.1 ml PLA s o l u t i o n ( t r e a t e d as m e n t i o n e d above) were i n -c l u d e d i n t h e r e a c t i o n m i x t u r e . T h i s m i x t u r e was i n c u b a t e d a t 37°C f o r 15 m i n u t e s , t h e n t h e r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f an e q u a l volume o f c o l d 4.0 mM EDTA pH 7.0. The membranes were c e n t r i f u g e d a t 40,000 x g f o r 5 m i n u t e s , washed once w i t h 20 mM T r i s pH 8.0, and r e s u s p e n d e d i n c o l d d o u b l e d i s t i l l e d w a t e r t o a p p r o x i m a t e l y t h e i n i t i a l volume. C o n t r o l membranes were t r e a t e d s i m i l a r l y e x c e p t t h a t w a t e r was s u b s t i t u t e d f o r t h e PLA s o l u t i o n . ENZYME ASSAYS a) M a g n e s i u m - s t i m u l a t e d and P o t a s s i u m - s t i m u l a t e d N i t r o -p h e n y l p h o s p h a t a s e The e f f e c t s o f d i v a l e n t c a t i o n s on t h e enzyme n i t r o -p h e n y l p h o s p h a t a s e (NPPase) were s t u d i e d i n two d i f f e r e n t 2 + ways: i ) by s u b s t i t u t i n g t h e v a r i o u s d i v a l e n t c a t i o n s (M ) f o r t h e magnesium n o r m a l l y u s e d i n t h e a s s a y , and i i ) by s t u d y i n g t h e e f f e c t s o f v a r i o u s d i v a l e n t c a t i o n s i n t h e 2+ 2+ p r e s e n c e o f magnesium. B o t h t o t a l and Mg ( o r M ) NPPase were d e t e r m i n e d e x p e r i m e n t a l l y , w i t h K +-NPPase e s t i m a t e d as t h e d i f f e r e n c e between a c t i v i t i e s m e a s u r e d i n t h e p r e s e n c e and a b s e n c e o f K +. i ) S u b s t i t u t i o n o f M 2 + f o r M g 2 + D i v a l e n t c a t i o n - s t i m u l a t e d NPPase was d e t e r m i n e d i n a t o t a l volume o f 3.0 ml c o n t a i n i n g 1.0 ml 0.15 M 2 + i m i d a z o l e b u f f e r pH 7.4, 0.1 ml 30 mM M ( f i n a l c o n c e n t r a -t i o n 1 mM) as t h e c h l o r i d e s a l t s , 0.1 ml 0.09 M n i t r o p h e n y l -p h o s p h a t e , 0.2 ml membranes, and s u f f i c i e n t w a t e r t o make up t o 3.0 ml. T o t a l NPPase was d e t e r m i n e d s i m i l a r l y e x c e p t t h a t t h e r e a c t i o n m i x t u r e c o n t a i n e d 30 mM K +. C o n t r o l a c t i -v i t i e s i n t h e a b s e n c e o f d i v a l e n t c a t i o n s were a l s o d e t e r -mined. The r e a c t i o n was i n i t i a t e d by t h e a d d i t i o n o f t h e membranes, and t h e r e a c t i o n m i x t u r e was i n c u b a t e d w i t h s h a k i n g a t 37°C f o r one h o u r . A t t h e end o f t h i s t i m e , t h e r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f 1.0 ml c o l d 20% t r i c h l o r o a c e t i c a c i d (TCA). The s a m p l e s were c e n t r i f u g e d a t 40,000 x g f o r 5 m i n u t e s and a 3.0 ml a l i q u o t removed. To t h i s a l i q u o t was added 1.0 ml 1.5 M T r i s and t h e a b s o r b a n c e o f t h e r e s u l t i n g s o l u t i o n a t 412 nm was d e t e r m i n e d . The s p e c i f i c a c t i v i t y was c a l c u l a t e d f r o m t h e e g u a t i o n : S p e c i f i c a c t i v i t y = A b s o r b a n c e a t 412 nm 1 Q 3 _ 2.915 x mg p r o t e i n / t u b e nmoles/hour/mg p r o t e i n where 2.915 i s a c o n v e r s i o n f a c t o r p r e v i o u s l y d e t e r m i n e d f r o m a s t a n d a r d c u r v e o f n i t r o p h e n y l p h o s p h a t e v a l u e s . 2+ 2 + i i ) A d d i t i o n o f M w i t h Mg 2 + I n t h e s e e x p e r i m e n t s , e a c h sample f o r t h e Mg NPPase d e t e r m i n a t i o n c o n t a i n e d , i n a t o t a l volume o f 3.0 ml, 1.0 ml 0.15 i m i d a z o l e b u f f e r pH 7.4, 0.1 ml 0.09 M n i t r o -p h e n y l p h o s p h a t e , 0.1 ml 30 mM M g C l 2 , 0.1 ml 30 mM M 2 + ( f i n a l c o n c e n t r a t i o n 1 mM), 0.2 ml membranes and s u f f i c i e n t w a t e r t o make up t o 3.0 ml. Samples f o r t h e t o t a l NPPase d e t e r m i n a t i o n were s i m i l a r e x c e p t t h a t t h e r e a c t i o n m i x t u r e c o n t a i n e d 30 mM K +. I n c o n t r o l s f o r b o t h t h e t o t a l and M g 2 + - s t i m u l a t e d NPPase w a t e r was s u b s t i t u t e d f o r t h e 30 mM 2 + M s o l u t i o n s . The p r o c e d u r e f o l l o w e d was t h e same as t h a t d e s c r i b e d i n t h e p r e c e d i n g s e c t i o n . i b) A c e t y l c h o l i n e s t e r a s e The e f f e c t s o f d i v a l e n t c a t i o n s on a c e t y l c h o l i n e s t e r a s e a c t i v i t y were d e t e r m i n e d i n t h e f o l l o w i n g manner. E a c h sample ha d a t o t a l volume o f 3.0 ml c o n s i s t i n g o f 2.6 ml 0.1 M T r i s pH 8.0, 0.1 ml 0.01 M 5 , 5 1 - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c a c i d ) , 0.1 ml 0.0 3 M a c e t y l t h i o c h o l i n e c h l o r i d e , 0.1 ml 2 + 30 mM M and 0.1 ml membrane s u s p e n s i o n ( d i l u t e d 1/10 w i t h d o u b l e d i s t i l l e d w a t e r so t h e p r o t e i n c o n t e n t was 0.3-0.5 2 + mg/ml). C o n t r o l s were s i m i l a r e x c e p t t h e 30 mM M was s u b s t i t u t e d by T r i s b u f f e r . The r e a c t i o n was i n i t i a t e d w i t h t h e a d d i t i o n o f t h e a c e t y l t h i o c h o l i n e and t h e a b s o r b a n c e o f t h e s o l u t i o n was m easured a t 412 nm a t 30 s e c o n d i n t e r v a l s f o r lJf m i n u t e s . S l o p e s f o r t h e i n c r e a s e o f a b s o r b a n c e w i t h t i m e were d e t e r m i n e d and e x p r e s s e d as a p e r c e n t i n c r e a s e o r d e c r e a s e o f t h e c o n t r o l v a l u e . MISCELLANEOUS PROCEDURES a) I n c o r p o r a t i o n o f P i c r y l S u l p h o n i c A c i d and P i c r y l C h l o r i d e The i n c o r p o r a t i o n o f p i c r y l s u l p h o n i c a c i d (TNBS) i n t o e r y t h r o c y t e membranes s t i m u l a t e d by v a r i o u s d i v a l e n t and m o n o v a l e n t c a t i o n s was s t u d i e d as a f u n c t i o n o f t e m p e r a t u r e i n t h e f o l l o w i n g manner. R e a c t i o n m i x t u r e s c o n s i s t i n g o f 1.0 ml 20 mM T r i s pH 8.0, 0.1 ml c a t i o n s o l u t i o n (30 mM f o r d i v a l e n t i o n s , o r 300 mM f o r m o n o v a l e n t i o n s ) , 0.1 ml 10 mM TNBS pH 8.0, 0.2 ml membrane s u s p e n s i o n , and s u f f i c i e n t w a t e r t o make a f i n a l volume o f 3.0 ml, were i n c u b a t e d a t f i v e d i f f e r e n t t e m p e r a t u r e s c h o s e n between 5 and 4 6 ° C f o r 60 s e c o n d s ( d i v a l e n t i o n s ) o r 10 m i n u t e s ( m o n o v a l e n t i o n s ) . Two m i l l i l i t e r s o f a 1:1 m i x t u r e o f 1 M HC1:10% s o d i u m d o d e c y l s u l p h a t e (SDS) were u s e d to s t o p t h e r e a c t i o n and s o l u b i l i z e t h e membranes. The a b s o r b a n c e o f e a c h sample was m e a s u r e d a t 335 nm. Samples c o n t a i n i n g b a r i u m f o r m e d a p r e -c i p i t a t e i n t h e p r e s e n c e o f SDS w h i c h was removed by c e n t r i -f u g a t i o n b e f o r e d e t e r m i n a t i o n o f a b s o r b a n c e . The i n c o r p o r a t i o n o f p i c r y l c h l o r i d e (PC) s t i m u l a t e d by d i v a l e n t c a t i o n s was s t u d i e d by t h e same method, w i t h 10 mM PC ( d i s s o l v e d i n e t h a n o l ) r e p l a c i n g t h e 10 mM TNBS. C o n t r o l s f o r b o t h t h e TNBS and PC i n c o r p o r a t i o n were r u n i n t h e a b s e n c e o f d i v a l e n t o r m o n o v a l e n t c a t i o n s f o r r e a c t i o n t i m e s o f 30 m i n u t e s and 2 m i n u t e s r e s p e c t i v e l y . A r r h e n i u s p l o t s ( I n a b s o r b a n c e / m g p r o t e i n / m i n u t e a g a i n s t 1/T i n °K~^~) were c o n s t r u c t e d and s l o p e s and i n t e r -c e p t s o f t h e s e A r r h e n i u s p l o t s were d e t e r m i n e d by r e g r e s s i o n a n a l y s i s u s i n g a Compucorp 140 ( S t a t i s t i c i a n ) c a l c u l a t o r . T h ese v a l u e s were u s e d t o c a l c u l a t e a c t i v a t i o n e n e r g i e s ( E a = 1.9869 x s l o p e i n c a l . / m o l e ) and r a t e s o f r e a c t i o n ( e x p r e s s e d as I n absorbance/mg p r o t e i n / m i n u t e a t 2 5 ° C ) . A s e r i e s o f e x p e r i m e n t s was a l s o p e r f o r m e d t o d e t e r m i n e t h e e f f e c t s o f pH on t h e A r r h e n i u s p a r a m e t e r s o f TNBS and PC i n c o r p o r a t i o n . The method was t h e same as t h a t d e s c r i b e d above e x c e p t t h a t T r i s b u f f e r s o f v a r i o u s pH's ( f r o m 7.0 t o 9.0 i n 0.5 pH u n i t i n c r e m e n t s ) were u t i l i z e d , and t h e r e -a c t i o n t i m e s were v a r i e d f r o m 30 - 60 s e c o n d s , a t h i g h pH, t o 12 - 24 m i n u t e s , a t low pH. Magnesium was c h o s e n as a r e p r e s e n t a t i v e d i v a l e n t i o n and s o d i u m as a r e p r e s e n t a t i v e m o n o v a l e n t i o n i n t h e s e s t u d i e s . A p a r a l l e l s t u d y was a l s o c o n d u c t e d w i t h b u t y l a m i n e u s e d as a s i m p l e model amino com-2+ pound. U n s t i m u l a t e d TNBS and PC i n c o r p o r a t i o n and Mg s t i m u l a t e d TNBS i n c o r p o r a t i o n i n t o b u t y l a m i n e were s t u d i e d by e x a c t l y t h e same method as t h e i n c o r p o r a t i o n i n t o mem-b r a n e s , w i t h 0.2 ml b u t y l a m i n e ( d i l u t e d 1:500 w i t h d o u b l e d i s t i l l e d w a t e r ) r e p l a c i n g t h e 0.2 ml membranes. b) Column Chromatography o f Membranes L a b e l l e d W i t h TNBS i ) E r y t h r o c y t e Membranes Two m i l l i l i t e r s o f e r y t h r o c y t e membranes were l a b e l l e d w i t h TNBS i n a r e a c t i o n m i x t u r e c o n t a i n i n g 7.0 ml 20 mM T r i s pH 8.0, 0.7 ml 10 mM TNBS pH 8.0, 0.67 ml 30 mM 2 + M ( f i n a l c o n c e n t r a t i o n 1 mM - s u b s t i t u t e d by water i n the c o n t r o l s a m p l e ) , and s u f f i c i e n t water t o make a t o t a l volume of 20 ml. Samples were i n c u b a t e d a t 37°C f o r one hour, t h e r e a c t i o n s t opped by the a d d i t i o n o f 7.0 ml 1 M HC1, and the samples c e n t r i f u g e d a t 40,000 x g f o r 10 minutes. The mem-branes were washed once by c e n t r i f u g a t i o n w i t h 10 ml 20 mM T r i s pH 8.0, resuspended i n 1.5 ml water and d i a l y z e d a g a i n s t 5 mM EDTA - 5 mM 2-me r c a p t o e t h a n o l , pH 7.5, a t 4°C f o r 70 hour s . S o l u b i l i z a t i o n o f the samples was o b t a i n e d by a d d i n g 0.4 ml 10% SDS and b o i l i n g f o r 10 minutes. 1.0 ml a l i q u o t s were a p p l i e d t o a 16 x 100 mm column o f Sephadex G-200 and e l u t e d w i t h a s o l u t i o n c o n t a i n i n g 1% SDS - 0.02% sodium a z i d e - 0.05 M ammonium b i c a r b o n a t e . F i f t y drops ( a p p r o x i -m a t e l y 1.2 ml) were c o l l e c t e d i n each f r a c t i o n a t a r a t e o f 15-20 seconds p e r drop. The absorbance a t 335 nm o f each f r a c t i o n was measured and a l i q u o t s were assayed f o r p r o t e i n and p h o s p h o l i p i d c o n t e n t . i i ) B ovine S y n a p t i c V e s i c l e s and P u r i f i e d Rat L i v e r Microsomes Column chromatography o f b o v i n e s y n a p t i c v e s i c l e s and p u r i f i e d r a t l i v e r microsomes was performed s i m i l a r l y to t he e r y t h r o c y t e s e x c e p t t h a t the quantity:«of membranes l a b e l l e d was o n l y 1.5 ml f o r each sample. The o t h e r con-s t i t u e n t s o f t h e r e a c t i o n m i x t u r e were t h e r e f o r e r e d u c e d a c c o r d i n g l y . c) T h i n L a y e r Chromatography C a t i o n e f f e c t s on the l a b e l l i n g o f i n d i v i d u a l phos-p h o l i p i d s were a n a l y z e d by t h i n l a y e r chromatography. One m i l l i l i t e r o f e r y t h r o c y t e membranes was l a b e l l e d a t 37°C f o r 30 minutes i n a t o t a l volume o f 15 ml c o n s i s t i n g o f 5.0 ml 20 mM T r i s pH 8.0, 0.5 ml 10 mM TNBS pH 8.0, 0.5 ml 2+ 30 mM M and s u f f i c i e n t w a t e r to b r i n g t h e volume t o 15 ml L a b e l l e d and u n l a b e l l e d membrane c o n t r o l s were p r e p a r e d by the same method, s u b s t i t u t i n g w a t e r f o r the c a t i o n s o l u t i o n i n one case and f o r b o t h the c a t i o n s o l u t i o n and the TNBS i n the o t h e r . R e a c t i o n s were t e r m i n a t e d by the a d d i t i o n o f 5.0 ml 1 M HCI, the membranes c e n t r i f u g e d a t 40,000 x g f o r 10 m i n u t e s , and washed once w i t h 5.0 ml 20 mM T r i s pH 8.0. T h i s was f o l l o w e d by two e x t r a c t i o n s o f the membranes w i t h 2.0 ml c h l o r o f o r m i m e t h a n o l (2:1, v : v ) . The combined ex-t r a c t s were washed t h r e e times w i t h 1.0 ml 0.75% sodium c h l o r i d e and e v a p o r a t e d t o ne a r d r y n e s s under n i t r o g e n . The r e s i d u e was r e d i s s o l v e d i n 0.5 ml c h l o r o f o r m : m e t h a n o l (2:1) and 40 - 6 0 y u l a l i q u o t s were a p p l i e d t o a c t i v a t e d (30 minutes a t 110°C) s i l i c a g e l F254 p l a t e s (0.25 mm t h i c k n e s s , Brinkman). P h o s p h o l i p i d s were r e s o l v e d u s i n g c h l o r o -form : methanol : ammonia (14:6:1, v:v:v) as s o l v e n t system. The amino p h o s p h o l i p i d s p h o s p h a t i d y l e t h a n o l a m i n e and phos-p h a t i d y l s e r i n e were l o c a t e d w i t h n i n h y d r i n s p r a y ; phospha-t i d y l c h o l i n e and s p h i n g o m y e l i n were l o c a t e d w i t h i o d i n e v a p o u r . E a c h p h o s p h o l i p i d s p o t was e x t r a c t e d f r o m t h e s i l i c a g e l t w i c e w i t h 1.0 ml m e t h a n o l , t h e n t h e e x t r a c t e v a p o r a t e d u n d e r n i t r o g e n and a s s a y e d f o r p h o s p h a t e i n t h e u s u a l manner. d) I n c o r p o r a t i o n o f 5 , 5 1 - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c a c i d ) . The e f f e c t s o f d i v a l e n t c a t i o n s on membrane s u l f h y d r y l g r o u p r e a c t i v i t y were a n a l y z e d by s t u d y i n g t h e i n c o r p o r a t i o n o f 5 , 5 ' - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB). E a c h sample c o n t a i n e d 1.0 ml 0.15M i m i d a z o l e pH 7.4, 0.1 ml 0.3 mM DTNB ( d i s s o l v e d i n i m i d a z o l e b u f f e r ) , 0.1 ml 30 mM M 2 + , 0.2 ml membranes and s u f f i c i e n t w a t e r t o make a t o t a l volume o f 3.0 ml. I n c o n t r o l s , 0.1 ml w a t e r was s u b s t i t u t e d f o r t h e c a t i o n s o l u t i o n . The s a m p l e s were i n c u b a t e d a t 37°C f o r 30 m i n u t e s , c e n t r i f u g e d a t 40,000 x g and t h e a b s o r b a n c e o f t h e s u p e r n a t a n t a t 412 nm was d e t e r m i n e d . The number o f a c c e s s i b l e s u l f h y d r y l g r o u p s r e l a t i v e t o t h e c o n t r o l was c a l c u l a t e d u s i n g a m o l a r e x t i n c t i o n c o e f f i c i e n t o f 1.36 x 1 0 4 . (47) e) H e m o l y s i s The e f f e c t s o f d i v a l e n t and m o n o v a l e n t c a t i o n s on h y p o -t o n i c a l l y i n d u c e d h e m o l y s i s were t e s t e d u s i n g t h e f o l l o w i n g p r o c e d u r e . F i v e m i l l i l i t e r s o f f r e s h human b l o o d were t a k e n f r o m h e a l t h y v o l u n t e e r s u s i n g 50 u n i t s h e p a r i n / m l b l o o d as a n t i c o a g u l a n t . The b l o o d was c e n t r i f u g e d a t 4°C f o r 5 m i n -u t e s a t t o p s p e e d i n a c l i n i c a l c e n t r i f u g e and t h e p l a s m a and b u f f y c o a t removed. The p a c k e d r e d b l o o d c e l l s were w a s h e d t h r e e t i m e s w i t h f o u r v o l u m e s o f 0.25 M s u c r o s e -15 mM T r i s pH 7.0 a n d r e s u s p e n d e d i n 32 ml o f t h i s w a s h i n g medium. The c a t i o n s u n d e r s t u d y w ere d i s s o l v e d a t t h e d e -2+ + s i r e d c o n c e n t r a t i o n s (1 mM M , 10 mM M ) i n 0.15 M s u c r o s e -15 mM T r i s pH 7.0. To 1.5 ml o f e a c h c a t i o n s o l u t i o n was a d d e d 0.2 ml o f t h e r e d b l o o d c e l l s u s p e n s i o n . The s a m p l e s w e re i m m e d i a t e l y v o r t e x e d a n d i n c u b a t e d a t room t e m p e r a t u r e f o r 15 m i n u t e s . A t t h e end o f t h i s t i m e , c e l l s w e r e s u b -j e c t e d t o a h y p o t o n i c c h a l l e n g e b y t h e a d d i t i o n o f 2.3 ml 2+ + o f 15 mM T r i s pH 7.0 c o n t a i n i n g 1 mM M o r 10 mM M . The s o l u t i o n s w e r e v o r t e x e d and i n c u b a t e d 10 m i n u t e s a t room t e m p e r a t u r e t h e n c e n t r i f u g e d a t 40,000 x g f o r 1 m i n u t e . S u p e r n a t a n t s w e r e r e m o v e d and t h e a b s o r b a n c e s d e t e r m i n e d a t 540 nm. A l l e x p e r i m e n t s w e r e p e r f o r m e d i n t r i p l i c a t e a n d r e s u l t s e x p r e s s e d r e l a t i v e t o c o n t r o l v a l u e s (where no c a t i o n s w e r e a d d e d ) . f ) S o n i c a t i o n E r y t h r o c y t e membranes were s o n i c a t e d i n t h e p r e s e n c e o f 2+ + 1 mM M o r 10 mM M by t h e f o l l o w i n g metnod. 1.3 ml mem-2+ + b r a n e s were m i x e d w i t h 0.2 ml M s o l u t i o n (7.5 mM) o r M s o l u t i o n (75 mM) o r w a t e r ( c o n t r o l ) . The s u s p e n s i o n was s o n i c a t e d i n i c e f o r a t o t a l o f 2 m i n u t e s a c t u a l s o n i c a t i o n t i m e ( i n 15 s e c o n d i n t e r v a l s s e p a r a t e d b y 15 ^ s e c o n d c o o l i n g i n t e r v a l s ) u s i n g a B r a u n 1510 s o n i c a t o r a t 50 w a t t s p ower. The a b s o r b a n c e o f t h e s o n i c a t e d s u s p e n s i o n was t h e n d e t e r -m i n e d a t 900 nm. S i m i l a r e x p e r i m e n t s w e r e c o n d u c t e d o n e r y t h r o c y t e membranes t r e a t e d w i t h t r y p s i n , n e u r a m i n i d a s e , o r PLA w i t h t h e e x c e p t i o n t h a t t h e n e u r a m i n i d a s e t r e a t e d membranes w e r e s o n i c a t e d f o r a t o t a l o f 4 m i n u t e s i n s t e a d o f t h e u s u a l 2 m i n u t e s . R a t l i v e r m i c r o s o m e s , p u r i f i e d r a t l i v e r m i c r o s o m e s , a n d b o v i n e s y n a p t i c v e s i c l e s w e r e a l s o s u b j e c t e d t o s o n i c a t i o n i n t h e same manner, w i t h t h e s o n i c a t i o n t i m e s b e i n g 4 m i n u t e s f o r t h e m i c r o s o m e s a n d 2 m i n u t e s f o r t h e s y n a p t i v e s i c l e s a n d p u r i f i e d m i c r o s o m e s . RESULTS The m a j o r o b j e c t i v e o f t h i s work was t o c h a r a c t e r i z e i n d e t a i l t h e membrane p e r t u r b a t i o n a l e f f e c t s o f i n o r g a n i c c a t i o n s u s i n g t h e human e r y t h r o c y t e membrane as a m o d e l s y s t e m and e m p l o y i n g a v a r i e t y o f p h y s i c o c h e m i c a l a n d b i o -c h e m i c a l t e c h n i q u e s . A C TIVATION ENERGY AND RATE STUDIES a) M o d e l S t u d i e s W i t h B u t y l a m i n e One o f t h e e a r l i e s t o f t h e s e a p p r o a c h e s was an exam-i n a t i o n o f t h e e f f e c t o f c a t i o n s o n t h e a c t i v a t i o n e n e r g y and r a t e o f i n c o r p o r a t i o n o f t h e amino g r o u p - s p e c i f i c p r o b e , t r i n i t r o b e n z e n e s u l f o n i c a c i d ( T N B S ) , i n t o e r y t h r o c y t e mem-b r a n e s . I n o r d e r t o i n t e r p r e t t h e d a t a o b t a i n e d w i t h t h i s p r o b e i n t e r m s o f membrane s t r u c t u r a l a l t e r a t i o n s i n d u c e d b y m o n o v a l e n t and d i v a l e n t c a t i o n s , i t was f i r s t n e c e s s a r y t o d i s t i n g u i s h d i r e c t e f f e c t s o f c a t i o n s o n amino g r o u p r e -a c t i v i t y f r o m i n d i r e c t e f f e c t s on r e a c t i v i t y a n d / o r a c c e s s i -b i l i t y a r i s i n g f r o m i o n - i n d u c e d membrane a l t e r a t i o n s . To t h i s e n d , t h e i n c o r p o r a t i o n o f TNBS i n t o a s i m p l e m o d e l amino compound, b u t y l a m i n e , was i n v e s t i g a t e d b o t h i n t h e a b s e n c e o f c a t i o n s a n d i n t h e p r e s e n c e o f r e p r e s e n t a t i v e 2+ + d i v a l e n t (Mg ) and m o n o v a l e n t (Na ) c a t i o n s . I t was a l s o i m p o r t a n t t o s e p a r a t e t h o s e e f f e c t s o f c a t i o n s o n t h e i n -c o r p o r a t i o n o f t h e a n i o n i c TNBS m o l e c u l e a r i s i n g f r o m s i m p l e c h a r g e n e u t r a l i z a t i o n f r o m t h o s e i n v o l v i n g i o n - d e p e n d e n t s t r u c t u r a l p e r t u r b a t i o n s . E x p e r i m e n t s u t i l i z i n g p i c r y l c h l o r i d e ( P C ) , a n e u t r a l a n a l o g u e o f TNBS w h i c h g e n e r a t e s t h e same t r i n i t r o p h e n y l a t e d amino d e r i v a t i v e as TNBS, were p a r t i c u l a r l y e n l i g h t e n i n g i n t h i s r e g a r d . F i g u r e s 1 and 2 show t h e a c t i v a t i o n e n e r g i e s and t h e r a t e s o f i n c o r p o r a t i o n a t 25°C as a f u n c t i o n o f pH f o r t h e r e a c t i o n o f TNBS and PC w i t h e r y t h r o c y t e membranes. F o r b o t h p r o b e s , t h e r a t e o f i n c o r p o r a t i o n d e c r e a s e d w i t h d e -c r e a s i n g pH, w i t h PC b e i n g i n c o r p o r a t e d i n t o t h e membranes a t a h i g h e r r a t e t h a n TNBS a t a l l pH v a l u e s s t u d i e d . I n t h e c a s e o f PC, t h e d e c r e a s i n g r a t e o f i n c o r p o r a t i o n was a s s o c i a t e d w i t h an i n c r e a s i n g a c t i v a t i o n e n e r g y , as i s t o be e x p e c t e d . However, w i t h TNBS, t h e d e c r e a s e i n r a t e o f i n c o r p o r a t i o n as pH was l o w e r e d was a s s o c i a t e d w i t h a p r o - . g r e s s i v e d e c r e a s e i n a c t i v a t i o n e n e r g y , a f i n d i n g t h a t was u n e x p e c t e d f o r a s u p p o s e d l y s t r a i g h t f o r w a r d amino g r o u p -p r o b e r e a c t i o n . The r e s u l t s o f an a n a l o g o u s s e r i e s o f e x p e r i m e n t s w i t h b u t y l a m i n e a r e shown i n F i g u r e s 3 and 4. As w i t h e r y t h r o c y t e membranes, t h e a c t i v a t i o n e n e r g y i n c r e a s e d and t h e r a t e o f i n c o r p o r a t i o n a t 25°C d e c r e a s e d w i t h d e c r e a s i n g pH f o r t h e p r o b e PC. S i m i l a r r e s u l t s were a l s o o b t a i n e d f o r TNBS, a l -t h o u g h i t was o b s e r v e d t h a t t h e r e a c t i v i t y o f amino g r o u p s w i t h TNBS, as r e f l e c t e d by t h e r a t e o f i n c o r p o r a t i o n a t 25°C, was g r e a t e r t h a n t h a t w i t h PC a t a l l pH v a l u e s s t u d i e d , a s i t u a t i o n w h i c h was o p p o s i t e t o t h a t f o u n d i n e r y t h r o c y t e 32a F i g u r e 1 A c t i v a t i o n E n e r g i e s f o r t h e I n c o r p o r a t i o n o f TNBS and PC i n t o E r y t h r o c y t e Membranes as a F u n c t i o n o f pH TNBS and PC i n c o r p o r a t i o n were measured a t f i v e d i f f e r e n t t e m p e r a t u r e s i n t h e r a n g e 5-46°C f o r e a c h pH v a l u e , as d e s c r i b e d i n t h e Methods s e c t i o n . The d a t a a r e p r e s e n t e d i n t h e f o r m o f A r r h e n i u s p l o t s w i t h a c t i v a t i o n e n e r g i e s c a l c u l a t e d by m u l t i p l y i n g t h e s l o p e s o f t h e s e A r r h e n i u s p l o t s by t h e gas c o n s t a n t R. FIGURE 1 7.0 7.5 S.O Q,5 g.a p H 3 3a F i g u r e 2 R a t e s o f I n c o r p o r a t i o n a t 25°C o f TNBS and PC i n t o E r y t h r o c y t e Membranes as a F u n c t i o n o f pH TNBS and PC i n c o r p o r a t i o n were e v a l u a t e d as f o r F i g u r e 1, and t h e r a t e s o f i n c o r p o r a t i o n a t 25°C c a l c u l a t e d f r o m t h e s l o p e s and i n t e r c e p t s o f t h e A r r h e n i u s p l o t s . 3 3b FIGURE 2 | ° — — I » . L . ; : L _ 7 . 0 7.5 S . O S . 5 9 . 0 p H 34a F i g u r e 3 A c t i v a t i o n E n e r g i e s f o r t h e I n c o r p o r a t i o n o f TNBS and PC i n t o B u t y l a m i n e as a F u n c t i o n o f pH TNBS and PC i n c o r p o r a t i o n were measured a t f i v e d i f f e r e n t t e m p e r a t u r e s i n t h e r a n g e 5-46°C f o r e a c h pH v a l u e , as d e s c r i b e d i n t h e Methods s e c t i o n , t h e d a t a e x p r e s s e d i n t h e f o r m o f A r r h e n i u s p l o t s , and a c t i v a t i o n e n e r g i e s c a l c u l a t e d by m u l t i p l y i n g t h e s l o p e s o f t h e s e A r r h e n i u s p l o t s by t h e gas c o n s t a n t R. 34b FIGURE 3 35a F i g u r e 4 R a t e s o f I n c o r p o r a t i o n a t 25^C o f TNBS and PC i n t o B u t y l a m i n e as a F u n c t i o n o f pH TNBS and PC i n c o r p o r a t i o n w e r e a n a l y z e d a s i n F i g u r e 3, and t h e r a t e s o f i n c o r p o r a t i o n a t 25°C c a l c u l a t e d f r o m t h e s l o p e s a n d i n t e r c e p t s o f t h e A r r h e n i u s p l o t s . 35b FIGURE 4 membranes. T h i s l a t t e r o b s e r v a t i o n i n d i c a t e d t h a t t h e i n -c r e a s e d r a t e o f i n c o r p o r a t i o n o f PC i n t o e r y t h r o c y t e mem-b r a n e s r e l a t i v e t o TNBS ( F i g u r e 2) was n o t a t t r i b u t a b l e t o a g r e a t e r i n t r i n s i c r e a c t i v i t y o f t h e m o l e c u l e a n d s u g g e s t e d t h a t i n c r e a s e d p e r m e a t i o n o f t h e u n c h a r g e d PC was p r o b a b l y r e s p o n s i b l e f o r t h e g r e a t e r r a t e o f i n c o r p o r a t i o n i n t o mem-b r a n e s . The i n c r e a s e d a c t i v a t i o n e n e r g y and d e c r e a s e d r a t e o f i n c o r p o r a t i o n o f PC and TNBS i n t o b u t y l a m i n e w i t h d e -c r e a s i n g pH w e r e a t t r i b u t e d t o p r o g r e s s i v e c o n v e r s i o n o f t h e amino g r o u p s o f t h e s u b s t r a t e t o t h e i r c h e m i c a l l y l e s s r e a c t i v e p r o t o n a t e d f o r m s . S i n c e i n o r g a n i c c a t i o n s w e re shown t o m a r k e d l y i n c r e a s e i n c o r p o r a t i o n o f TNBS i n t o e r y t h r o c y t e membranes ( a s w i l l be d e s c r i b e d s h o r t l y ) , e x p e r i m e n t s w i t h d i v a l e n t a n d m o n o v a l e n t 2+ + c a t i o n s , r e p r e s e n t e d b y Mg and Na , were p e r f o r m e d u s i n g b u t y l a m i n e i n o r d e r t o d e t e r m i n e w h e t h e r t h e s e i o n s a f f e c t e d amino g r o u p r e a c t i v i t y d i r e c t l y . A t t h e c o n c e n t r a t i o n s 2+ + s t u d i e d (ImM f o r Mg , 10 mM f o r Na ), m i n i m a l e f f e c t s w e re n o t e d , w i t h s l i g h t i n c r e a s e s i n r a t e a t t r i b u t a b l e t o i n -c r e a s e d i o n i c s t r e n g t h . W i t h e r y t h r o c y t e membranes, how-e v e r , p r o n o u n c e d c h a n g e s i n a c t i v a t i o n e n e r g i e s a n d r a t e s o f 2+ 2+ i n c o r p o r a t i o n w e r e o b s e r v e d w i t h b o t h Mg and Na . I t seemed l i k e l y t h a t t h e s e v a r i a t i o n s w e re t h e r e s u l t o f mem-b r a n e p e r t u r b a t i o n s a l t e r i n g t h e p e r m e a b i l i t y o f t h e p r o b e a n d / o r t h e a c c e s s i b i l i t y o f t h e amino g r o u p s . b ) E r y t h r o c y t e Membrane S t u d i e s F u r t h e r s t u d i e s were c o n d u c t e d w i t h v a r i o u s d i v a l e n t and monovalent c a t i o n s t o d e t e r m i n e t h e i r e f f e c t s on TNBS i n c o r p o r a t i o n i n t o e r y t h r o c y t e membranes. The degree o f TNBS i n c o r p o r a t i o n was d e t e r m i n e d a t s e v e r a l t e m p e r a t u r e s i n the pr e s e n c e and absence o f d i v a l e n t o r monovalent c a t i o n s and the r e s u l t s e x p r e s s e d i n the form o f A r r h e n i u s p l o t s ( F i g u r e 5) from which a c t i v a t i o n e n e r g i e s and r a t e s o f i n -c o r p o r a t i o n a t 25°C were de t e r m i n e d . These were then p l o t t e d a g a i n s t the c a t i o n s i z e ( i o n i c r a d i u s ( 4 8 ) ) . D i v a l e n t c a t i o n s were found to e x h i b i t a c e r t a i n degree o f s i z e dependence i n b o t h a c t i v a t i o n energy and r a t e o f i n c o r p o r a -t i o n a t 25°C ( F i g u r e 6 ) , u n l i k e the monovalent c a t i o n s which were independent of i o n i c r a d i u s ( F i g u r e 7 ) . Moreover, t h e a c t i v a t i o n energy and r a t e o f i n c o r p o r a t i o n a t 25°C demon-s t r a t e d d i f f e r e n t dependences on d i v a l e n t c a t i o n s i z e , s u g g e s t i n g t h a t t h e s e two e x p e r i m e n t a l q u a n t i t i e s may be measuring a t l e a s t p a r t i a l l y s e p a r a t e consequences o f c a t i o n -membrane i n t e r a c t i o n . I n t e r e s t i n g l y , the a c t i v a t i o n energy and r a t e p r o f i l e s f o r the v a r i o u s d i v a l e n t c a t i o n s t e s t e d q u a l i t a t i v e l y resembled the dependence o f f i r s t and second i o n i z a t i o n p o t e n t i a l s r e s p e c t i v e l y , on i o n i c r a d i u s ( F i g u r e 8 ( 4 9 ) ) . I t seemed p o s s i b l e , t h e r e f o r e , t h a t the p r o p e r -t i e s o f c a t i o n s t h a t d e t e r m i n e t h e i r i o n i z a t i o n p o t e n t i a l s may be r e l e v a n t t o t h e i r membrane-perturbing p r o p e r t i e s as r e f l e c t e d i n r a t e and a c t i v a t i o n energy parameters. S i n c e TNBS e x i s t s p r e d o m i n a n t l y as a n e g a t i v e l y c h a r g e d i o n a t the pH a t which the c a t i o n s t u d i e s were c a r r i e d o u t , i t was c o n s i d e r e d p o s s i b l e t h a t the i n c r e a s e d i n c o r p o r a t i o n 38a F i g u r e 5 A r r h e n i u s P l o t s f o r t h e I n c o r p o r a t i o n o f TNBS i n t o E r y t h r o c y t e Membranes i n t h e Ab s e n c e o r P r e s e n c e o f M o n o v a l e n t o r D i v a l e n t C a t i o n s TNBS i n c o r p o r a t i o n was d e t e r m i n e d a t f i v e d i f f e r e n t t e m p e r a t u r e s i n t h e r a n g e 5-46°C u s i n g r e a c t i o n t i m e s o f 30 m i n u t e s , 10 m i n u t e s , and 1 m i n u t e f o r + 2 + c o n t r o l , 10 mM K , and 1 mM Co r e s p e c t i v e l y . D u p l i c a t e d e t e r m i n a t i o n s were a l w a y s p e r f o r m e d . The d a t a were a n a l y z e d u s i n g a Compucorp 140 s t a t i s t i c a l c a l c u l a t o r t o e v a l u a t e s l o p e s and i n t e r c e p t s f r o m w h i c h a c t i v a t i o n e n e r g i e s and r a t e s o f i n c o r p o r a t i o n a t 25°C were c a l c u l a t e d . 38b FIGURE 5 39a F i g u r e 6 A c t i v a t i o n E n e r g i e s and R a t e s o f I n c o r p o r a t i o n a t 25°C f o r TNBS I n c o r p o r a t i o n i n t o E r y t h r o c y t e Membranes i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s TNBS i n c o r p o r a t i o n was d e t e r m i n e d f o r e a c h d i v a l e n t c a t i o n (1 mM f i n a l c o n c e n t r a t i o n ) as d e s c r i b e d f o r F i g u r e 5 a n d a c t i v a t i o n e n e r g i e s a n d r a t e s o f i n c o r p o r a t i o n a t 25°C were t h e n e v a l u a t e d f r o m t h e A r r h e n i u s p l o t s . E a c h p o i n t r e p r e s e n t s t h e mean - s t a n d a r d e r r o r o f t h e mean o f d u p l i c a t e d e t e r m i n a t i o n s on t h r e e d i f f e r e n t membrane p r e -p a r a t i o n s . FIGURE 6 40a F i g u r e 7 A c t i v a t i o n E n e r g i e s and R a t e s o f I n c o r p o r a t i o n a t 25°C f o r TNBS I n c o r p o r a t i o n i n t o E r y t h r o c y t e Membranes i n t h e P r e s e n c e o f M o n o v a l e n t C a t i o n s TNBS i n c o r p o r a t i o n was d e t e r m i n e d f o r e a c h mono-v a l e n t c a t i o n (10 mM f i n a l c o n c e n t r a t i o n ) as d e s c r i b e d f o r F i g u r e 5 and a c t i v a t i o n e n e r g i e s and r a t e s o f i n c o r p o r a t i o n a t 25°C were t h e n e v a l u a t e d f r o m t h e A r r h e n i u s p l o t s . E a c h p o i n t r e p r e s e n t s t h e mean - s t a n d a r d e r r o r o f t h e mean o f d u p l i c a t e d e t e r m i n a t i o n s on t h r e e d i f f e r e n t membrane p r e -p a r a t i o n s . 40b FIGURE 7, 6 N a R b [ UNSTIMULATED = 7 . 2 B ± 0 . 5 a ] ' ' " ' N a [ UIMSTIMUL ATEO = -B. HS ± D . i o ] 4> C s T • R b + .B I.O US 1 . 6 I.B C R Y S T A L R A D I U S ( A ) 4 l a F i g u r e 8 F i r s t and Second I o n i z a t i o n P o t e n t i a l s o f D i v a l e n t C a t i o n s V a l u e s f o r f i r s t and s e c o n d i o n i z a t i o n p o t e n t i a l s o f d i v a l e n t c a t i o n s were o b t a i n e d f r o m r e f e r e n c e 49 and p l o t t e d a g a i n s t c a t i o n i o n i c r a d i u s i n t h e same manner as f o r p r e c e d i n g g r a p h s . FIGURE 8 > J I S -< Z h • < N 1 3 ' i • " 2 • U LO i s t i . P . i c H I. P . IM i • I I f I I I I » ; M n f . S I .O T 1 . 2 C R Y S T A L R A D I U S ( o f TNBS i n t h e p r e s e n c e o f c a t i o n s c o u l d i n v o l v e a c o n t r i -b u t i o n f r o m c h a r g e n e u t r a l i z a t i o n o r s c r e e n i n g , e s p e c i a l l y s i n c e t h e e x t e r n a l s u r f a c e o f e r y t h r o c y t e membranes i s known to c o n t a i n many n e g a t i v e l y c h a r g e d g r o u p s , d e r i v e d m a i n l y f r o m s i a l i c a c i d , w h i c h c o u l d r e p e l t h e a n i o n i c TNBS m o l e -c u l e . I n o r d e r t o t e s t t h i s p o s s i b i l i t y , a s e r i e s o f ex-p e r i m e n t s was c o n d u c t e d on e r y t h r o c y t e membranes t r e a t e d w i t h n e u r a m i n i d a s e , r e s u l t i n g i n r e m o v a l o f a p p r o x i m a t e l y 70% o f t h e membrane s i a l i c a c i d . TNBS i n c o r p o r a t i o n i n t o t h e s e t r e a t e d membranes and i n t o c o n t r o l membranes was s t u d i e d as a f u n c t i o n o f pH i n t h e a b s e n c e and p r e s e n c e o f 2 + d i v a l e n t and m o n o v a l e n t c a t i o n s , r e p r e s e n t e d by Mg and N a + ( F i g u r e 9 ) . No d i f f e r e n c e s were n o t e d between c o n t r o l and n e u r a m i n i d a s e - t r e a t e d membranes w i t h r e s p e c t t o a c t i -v a t i o n e n e r g i e s o r r a t e s o f i n c o r p o r a t i o n i n e i t h e r t h e p r e s e n c e o r a b s e n c e o f c a t i o n s , s u g g e s t i n g t h a t r e p u l s i o n o f t h e TNBS m o l e c u l e by n e g a t i v e l y c h a r g e d s i a l i c a c i d r e s i d u e s i s u n l i k e l y t o be an i m p o r t a n t f a c t o r i n d e t e r -m i n i n g a c t i v a t i o n e n e r g i e s o r r a t e s o f i n c o r p o r a t i o n . A more d i r e c t a p p r o a c h t o t h e r o l e o f p r o b e c h a r g e i n d e t e r m i n i n g c a t i o n e f f e c t s i n v o l v e d t h e u s e o f p i c r y l c h l o r i d e ( P C ) , a n e u t r a l t r i n i t r o p h e n y l a t i n g a n a l o g u e o f TNBS. I t was f o u n d t h a t a l l t h e d i v a l e n t c a t i o n s , w i t h t h e 2 + e x c e p t i o n o f N i , p r o d u c e d e s s e n t i a l l y t h e same s m a l l d e -c r e a s e i n a c t i v a t i o n e n e r g y f r o m t h e c o n t r o l v a l u e ( F i g u r e 1 0 ) . T hese f i n d i n g s , w h i c h c l e a r l y d i f f e r e d f r o m t h e r e s u l t s o f s i m i l a r e x p e r i m e n t s w i t h TNBS ( F i g u r e 6) s u g -43a F i g u r e 9 A c t i v a t i o n E n e r g i e s and Rates o f I n c o r p o r a t i o n a t 25°C f o r TNBS I n c o r p o r a t i o n i n t o U n t r e a t e d and Neuraminidase T r e a t e d E r y t h r o c y t e Membranes i n the Absence o r Presence o f a D i v a l e n t o r Mono-v a l e n t C a t i o n Membranes were t r e a t e d w i t h n e u r a m i n i d a s e and C a C ^ i n sodium a c e t a t e b u f f e r (pH 5.5) as des-c r i b e d i n the Methods s e c t i o n . C o n t r o l membranes were t r e a t e d s i m i l a r l y e x c e p t the n e u r a m i n i d a s e was o m i t t e d . TNBS i n c o r p o r a t i o n was d e t e r m i n e d as d e s c r i b e d i n the Methods s e c t i o n i n the absence o f added c a t i o n o r i n the p r e s e n c e o f a r e p r e -2 + s e n t a t i v e d i v a l e n t (Mg , 1 mM f i n a l c o n c e n t r a t i o n ) o r monovalent ( N a + , 10 mM f i n a l c o n c e n t r a t i o n ) c a t i o n . A c t i v a t i o n e n e r g i e s and r a t e s o f i n c o r -p o r a t i o n a t 25°C were then e v a l u a t e d as d e s c r i b e d f o r F i g u r e 5. 43b FIGURE 9 P H 44a F i g u r e 10 A c t i v a t i o n E n e r g i e s and R a t e s o f I n c o r p o r a t i o n a t 25°C f o r PC I n c o r p o r a t i o n i n t o E r y t h r o c y t e Membranes i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s PC i n c o r p o r a t i o n i n t h e p r e s e n c e o f d i v a l e n t c a t i o n s (1 mM f i n a l c o n c e n t r a t i o n ) was d e t e r -m i n e d as d e s c r i b e d i n t h e Methods s e c t i o n . The d a t a a r e p r e s e n t e d i n t h e f o r m o f A r r h e n i u s p l o t s and a c t i v a t i o n e n e r g i e s and r a t e s o f i n -c o r p o r a t i o n a t 25°C c a l c u l a t e d f r o m t h e s l o p e s and i n t e r c e p t s o f t h e s e p l o t s . E a c h p o i n t r e p r e -s e n t s t h e mean - s t a n d a r d e r r o r o f t h e mean o f d u p l i c a t e d e t e r m i n a t i o n s on t h r e e d i f f e r e n t membrane p r e p a r a t i o n s . 44b FIGURE 10 C R Y S T A L R A D I U S ( A ) g e s t e d t h a t the a c t i v a t i o n energy term might be p r i m a r i l y a measure o f membrane s t r u c t u r a l a l t e r a t i o n s a f f e c t i n g probe p e r m e a t i o n . The i n f l u e n c e of d i v a l e n t c a t i o n s on the r a t e o f PC i n c o r p o r a t i o n as compared w i t h TNBS i n c o r p o r a t i o n p r e s e n t e d a d i f f e r e n t p i c t u r e from t h a t seen w i t h a c t i v a t i o n energy f o r probe i n c o r p o r a t i o n . I t can be seen by comparing t h e r e s u l t s w i t h PC ( F i g u r e 10) w i t h t h o s e f o r TNBS ( F i g u r e 6 ) , 2 + t h a t , w i t h the e x c e p t i o n o f N i , t h e r e i s a re m a r k a b l e de-gree o f s i m i l a r i t y i n the s i z e dependence o f the r a t e of i n c o r p o r a t i o n o f b o t h probes. The anomalously low r a t e of 2+ i n c o r p o r a t i o n o f PC i n the pr e s e n c e o f N i may, i n some manner, be l i n k e d t o the u n e x p e c t e d l y h i g h a c t i v a t i o n energy, s u g g e s t i n g t h a t , d e s p i t e the d i f f e r e n c e i n s i z e de-pendence between a c t i v a t i o n energy and r a t e o f i n c o r p o r a -t i o n , t h e r e may be some o v e r l a p i n t y p e s o f membrane p e r -t u r b a t i o n s measured by the s e two e x p e r i m e n t a l q u a n t i t i e s . ROLE OF MEMBRANE LIPIDS AND PROTEINS a) Membrane M o d i f i c a t i o n by P r o t e o l y t i c o r P h o s p h o l i p a s e Enzymes In a t t e m p t i n g t o a n a l y z e the m o l e c u l a r b a s i s o f the f o r e g o i n g d i v a l e n t c a t i o n e f f e c t s on a c t i v a t i o n energy and r a t e , a major q u e s t i o n c e n t e r e d about the r o l e of membrane p r o t e i n s and l i p i d s i n d e t e r m i n i n g t h e s e e f f e c t s . One approach t o t h i s problem i n v o l v e d a s t u d y o f d i v a l e n t c a t i o n - s t i m u l a t e d TNBS i n c o r p o r a t i o n i n t o membranes p r e -v i o u s l y t r e a t e d w i t h p r o t e o l y t i c o r p h o s p h o l i p a s e enzymes. T r y p s i n i z a t i o n o f membranes r e s u l t e d i n i n c r e a s e d a c t i v a -t i o n e n e r g i e s i n t h e p r e s e n c e o f a l l d i v a l e n t c a t i o n s ex-2+ 2 + c e p t N i and Co ( F i g u r e 11, t o p ) . T r e a t m e n t o f membranes w i t h p h o s p h o l i p a s e A, however, l e f t a c t i v a t i o n e n e r g i e s u n -2+ 2+ 2+ c h a n g e d i n t h e p r e s e n c e o f Mg , Ca , and S r , and i n -c r e a s e d i n t h e p r e s e n c e o f o t h e r d i v a l e n t c a t i o n s . When r a t e s o f i n c o r p o r a t i o n a t 25°C were e x a m i n e d ( F i g u r e 11, b o t t o m ) , i t was f o u n d t h a t t r y p s i n i z a t i o n c a u s e d a d e c r e a s e d 2+ 2 + r a t e i n t h e p r e s e n c e o f N i and Co , b u t h a d no e f f e c t on r a t e s i n t h e p r e s e n c e o f o t h e r c a t i o n s . On t h e o t h e r hand, p h o s p h o l i p a s e A t r e a t m e n t d e c r e a s e d i n c o r p o r a t i o n i n t h e p r e s e n c e o f a l l i o n s , most n o t i c e a b l y w i t h t h e a l k a l i n e 2+ 2+ 2+ 2 + e a r t h i o n s Mg , Ca , S r , and Ba . These r e s u l t s t e n d t o s u g g e s t t h a t membrane p r o t e i n s p l a y a m a j o r r o l e i n d e t e r m i n i n g a c t i v a t i o n e n e r g i e s , w h i l e membrane p h o s p h o -l i p i d s a r e i m p o r t a n t i n d e t e r m i n i n g r a t e s o f i n c o r p o r a t i o n . M o r e o v e r , t h e r e w o u l d a p p e a r t o be v a r i a t i o n s among t h e i o n s as t o t h e r e l a t i v e i m p o r t a n c e o f e i t h e r membrane p r o t e i n s o r p h o s p h o l i p i d s i n d e t e r m i n i n g t h e i r e f f e c t s . b) C h e m i c a l C h a r a c t e r i z a t i o n o f L a b e l l e d Membranes S i n c e t h e d e g r e e o f p r o t e i n and p h o s p h o l i p i d p e r t u r -b a t i o n a p p e a r e d t o v a r y among t h e d i v a l e n t c a t i o n s , a more d e t a i l e d s t u d y o f t h e e f f e c t s o f d i v a l e n t c a t i o n s on TNBS i n c o r p o r a t i o n i n t o membrane p r o t e i n s and p h o s p h o l i p i d s was u n d e r t a k e n . Membranes were l a b e l l e d w i t h TNBS i n t h e p r e -s e n c e o r a b s e n c e o f d i v a l e n t c a t i o n s , s o l u b i l i z e d i n s o d i um 47 a F i g u r e 11 E f f e c t s o f Enzyme P r e t r e a t m e n t o f E r y t h r o c y t e Membranes on D i v a l e n t C a t i o n - S t i m u l a t e d TNBS I n c o r p o r a t i o n Membranes were p r e t r e a t e d w i t h t r y p s i n o r p h o s p h o l i p a s e A as d e s c r i b e d i n t h e Methods s e c t i o n . TNBS i n c o r p o r a t i o n i n t h e p r e s e n c e o f d i v a l e n t c a t i o n s (1 mM f i n a l c o n c e n t r a t i o n ) was measured i n t h e u s u a l way w i t h r e a c t i o n t i m e s o f 1 m i n u t e f o r t r y p s i n i z e d membranes and 2 m i n u t e s f o r p h o s p h o l i p a s e A t r e a t e d membranes. A r r h e n i u s p l o t s f o r p h o s p h o l i p a s e A t r e a t e d membranes e x h i b i t e d a d i s c o n t i n u i t y above 30°C, t h e r e f o r e t h e a c t i v a t i o n e n e r g i e s and r a t e s o f i n c o r p o r a t i o n shown h e r e were c a l c u l a t e d u s i n g o n l y t h e p o i n t s b e l o w and i n c l u d i n g 30°C. E a c h v a l u e r e p r e s e n t s t h e mean - s t a n d a r d e r r o r o f t h e mean o f e x p e r i -ments w i t h two d i f f e r e n t membrane p r e p a r a t i o n s . 4^ tr d o d e c y l s u l f a t e , and t h e p r o t e i n and p h o s p h o l i p i d components r e s o l v e d by g e l f i l t r a t i o n . T h i s method r e s u l t e d i n com-p l e t e s e p a r a t i o n o f t h e p r o t e i n components, w h i c h e l u t e d i n a complex p a t t e r n b e g i n n i n g a t t h e v o i d volume, f r o m t h e p h o s p h o l i p i d components, w h i c h e l u t e d much l a t e r , t h u s e n -a b l i n g c a t i o n e f f e c t s on p r o t e i n and p h o s p h o l i p i d components t o be s t u d i e d s e p a r a t e l y ( F i g u r e 1 2 ) . The d a t a f r o m t h e s e e x p e r i m e n t s i n d i c a t e d t h a t d i v a l e n t c a t i o n s i n c r e a s e t h e ex-t e n t o f TNBS i n c o r p o r a t i o n i n t o b o t h p r o t e i n and p h o s p h o l i p i d components, and t h a t t h e c a t i o n s d e m o n s t r a t e d i f f e r e n t a b i l i -t i e s t o p e r t u r b membrane p r o t e i n s and p h o s p h o l i p i d s . M a x i -mal TNBS i n c o r p o r a t i o n i n t o p r o t e i n s and p h o s p h o l i p i d s (as d e t e r m i n e d by t h e a b s o r b a n c e a t 335 nm) i n t h e p r e s e n c e o f e a c h c a t i o n i s shown as a f u n c t i o n o f i o n i c r a d i u s i n F i g u r e 13. C o n s i s t e n t w i t h e a r l i e r s p e c u l a t i o n s b a s e d on t h e ex-p e r i m e n t s w i t h t r y p s i n and p h o s p h o l i p a s e A r e g a r d i n g t h e r e l a t i v e i m p o r t a n c e o f p r o t e i n s and p h o s p h o l i p i d s i n d e t e r -m i n i n g a c t i v a t i o n e n e r g y and r a t e p a r a m e t e r s r e s p e c t i v e l y , i t was f o u n d t h a t t h e s i z e d e p e n d e n c e o f maximal TNBS i n -c o r p o r a t i o n i n t o t h e p h o s p h o l i p i d components s t r o n g l y r e -se m b l e s t h a t o f t h e r a t e o f i n c o r p o r a t i o n a t 25°C o f TNBS, w h i l e t h e r e i s a s i m i l a r c o r r e s p o n d e n c e between t h e s i z e d e p e n d e n c e o f maximal TNBS i n c o r p o r a t i o n i n t o t h e p r o t e i n components and t h e a c t i v a t i o n e n e r g y f o r TNBS i n c o r p o r a t i o n i n t o e r y t h r o c y t e membranes as a whole ( F i g u r e 6 ) . A more d e t a i l e d e x a m i n a t i o n o f t h e c a t i o n e f f e c t s on p h o s p h o l i p i d s by t h i n l a y e r c h r o m a t o g r a p h y i n d i c a t e d t h a t p h o s p h a t i d y l -49a F i g u r e 12 TNBS L a b e l l i n g P a t t e r n s o f E r y t h r o c y t e Membranes i n the Presence o r Absence o f D i v a l e n t C a t i o n s Membranes (2.0 ml) were l a b e l l e d w i t h TNBS i n the p r e s e n c e o r absence o f d i v a l e n t c a t i o n s (1 mM f i n a l c o n c e n t r a t i o n ) , washed, and s o l u -b i l i z e d as d e s c r i b e d i n the Methods s e c t i o n . 1.0 ml a l i q u o t s were a p p l i e d t o a 1.6 x 100 cm column o f Sephadex G-200 and e l u t e d w i t h 1% SDS - 0.0 2% sodium a z i d e - 0.05 M ammonium b i c a r b o n a t e . F i f t y drop f r a c t i o n s were c o l l e c t e d and the absorbance o f each f r a c t i o n measured a t 335 nm. UNSTIMULATED O O IV! g** A A M n _ ++• A—A C o 5 a •tr 6 a TO T O O FRACTION NO. 50a F i g u r e 13 Maximal TNBS L a b e l l i n g o f P r o t e i n and Phospho-l i p i d Components o f E r y t h r o c y t e Membranes i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s E x p e r i m e n t s were p e r f o r m e d as d e s c r i b e d f o r F i g u r e 12 and maximal a b s o r b a n c e s a t 335 nm o f t h e r e s o l v e d p r o t e i n and p h o s p h o l i p i d com-p o n e n t s d e t e r m i n e d f o r e a c h c a t i o n . E a c h p o i n t r e p r e s e n t s t h e mean o f e x p e r i m e n t s w i t h two d i f f e r e n t membrane p r e p a r a t i o n s . / FIGURE 13 e t h a n o l a m i n e was t h e m a j o r p h o s p h o l i p i d i n v o l v e d i n TNBS l a b e l l i n g , w i t h o n l y m i n o r e f f e c t s on t h e p h o s p h o l i p i d s p h o s p h a t i d y l s e r i n e , s p h i n g o m y e l i n , and p h o s p h a t i d y l c h o l i n e ( F i g u r e 1 4 ) . I t s h o u l d be n o t e d t h a t t h e s i z e d e p e n d e n c e o f d i v a l e n t c a t i o n - s t i m u l a t e d i n c o r p o r a t i o n o f TNBS i n t o t h e t o t a l p h o s p h o l i p i d f r a c t i o n ( F i g u r e 13) i s r e f l e c t e d i n t h e s i z e d e p e n d e n c e o f i n c o r p o r a t i o n i n t o p h o s p h a t i d y l -e t h a n o l a m i n e ( F i g u r e 1 4 ) . I t w o u l d a p p e a r , t h e r e f o r e , t h a t t h e membrane p e r t u r b a -t i o n a l e f f e c t s o f t h e d i v a l e n t c a t i o n s i n v o l v e a b a l a n c e b e -tween p r o t e i n and p h o s p h o l i p i d s t r u c t u r a l c h a n g e s . Based on t h i s , i t seemed l i k e l y t h a t c a t i o n s w h i c h i n d u c e d membrane p e r t u r b a t i o n s by t h e same mechanism w o u l d e x h i b i t a c o n s t a n t r e l a t i o n s h i p between p h o s p h o l i p i d and p r o t e i n l a b e l l i n g , e v e n t h o u g h t h e a c t u a l d e g r e e o f l a b e l l i n g i n t h e s e components m i g h t v a r y f r o m c a t i o n t o c a t i o n . The r a t i o s o f t h e maximal p h o s p h o l i p i d l a b e l l i n g t o t h e maximal p r o t e i n l a b e l l i n g were, t h e r e f o r e , d e t e r m i n e d f o r e a c h c a t i o n f r o m d a t a o b t a i n e d f r o m e x p e r i m e n t s s u c h as t h o s e shown i n F i g u r e 13, and t h e r e s u l t s s ummarized i n T a b l e I . P h o s p h o l i p i d components were a l w a y s l a b e l l e d more e x t e n s i v e l y t h a n p r o t e i n components, e v e n i n t h e c o n t r o l , so t h a t a l l r a t i o s a r e g r e a t e r t h a n 1. I n t h e p r e s e n c e o f c a t i o n s , t h e r e was a p r e f e r e n t i a l i n c r e a s e i n p h o s p h o l i p i d l a b e l l i n g , s u c h t h a t a l l r a t i o s w i t h c a t i o n s p r e s e n t a r e g r e a t e r t h a n t h e c o n t r o l v a l u e . I t c a n be s e e n 2 + 2 + f r o m T a b l e I t h a t t h e a l k a l i n e e a r t h c a t i o n s Mg , Ca , 2 + 2 + S r , and Ba t e n d t o f o r m a g r o u p w i t h r a t i o s o f a b o u t 5, 5 2a F i g u r e 14 M o d i f i c a t i o n o f E r y t h r o c y t e Membrane Phospho-l i p i d by TNBS i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s Membranes (2.0 ml) were l a b e l l e d w i t h TNBS and e x t r a c t e d as d e s c r i b e d i n t h e Methods s e c t i o n . I n d i v i d u a l p h o s p h o l i p i d s were d e t e r m i n e d by t h i n - l a y e r c h r o m a t o g r a p h y u s i n g c h l o r o f o r m / methanol/ammonia (14/6/1, v / v / v ) f o l l o w e d by p h o s p h a t e a n a l y s i s o f i n d i v i d u a l s p o t s e l u t e d f r o m t h e s i l i c a g e l w i t h m e t h a n o l . E a c h p o i n t r e p r e s e n t s t h e mean - s t a n d a r d e r r o r o f t h e mean f o r t h r e e d i f f e r e n t membrane p r e p a r a t i o n s . FIGURE 14 M rT P E T H A N O L A M I N E C a B a A — A P S E R I N E •---a S P H I N G O M Y E L I N 0 - - 0 P C H O L I N E C R Y S T A L R A D I U S ( A ) Table I . I n f l u e n c e of c a t i o n s on r e l a t i v e l a b e l l i n g o f e r y t h r o c y t e membrane p h o s p h o l i p i d s and p r o t e i n s by TNBS ,. .. maximal p h o s p h o l i p i d l a b e l l i n g  c a t l o n * r a t l ° maximal p r o t e i n l a b e l l i n g none Mg 2 + Ca 2 + Sr Ba 2 + 2 + Ni Co 2 + Mn 2 + 1.4 ± 0.2 5.6 ± 0.3 5.8 ± 0.2 5.3 ± 0.3 4.8 ± 0.4 1.8 ± 0.2 2.2 ± 0.1 3.4 ± 0.1 * D i v a l e n t c a t i o n s were t e s t e d a t a c o n c e n t r a t i o n o f 1 mM. These r e s u l t s r e p r e s e n t the mean ± SEM o b t a i n e d w i t h two d i f f e r e n t membrane p r e p a r a t i o n s . Membranes were l a b e l l e d and chromatographed as d e s c r i b e d f o r F i g . 12. Maximal p h o s p h o l i p i d and p r o t e i n l a b e l l i n g were e s t i m a t e d from the maximal absorbance a t 335 nm o f r e s o l v e d p h o s p h o l i p i d and p r o t e i n peaks f o r each i o n . while the other divalent cations Co i T, Ni^""1", and Mn',T, which have previously been found to possess marked protein per-turbing properties, form a heterogeneous group with much lower phospholipid to protein l a b e l l i n g r a t i o s . APPROACH TO FURTHER CHARACTERIZATION OF ION-INDUCED PROTEIN  PERTURBATIONS On the basis of the foregoing r e s u l t s , attention was focussed on the membrane protein perturbational character-i s t i c s of divalent cations and i n order to probe regions at l e a s t p a r t i a l l y d i s t i n c t from those possessing amino groups reactive towards TNBS, experiments were performed using 5,5 1-dithio-bis-(2-nitrobenzoic acid) (DTNB), a highly s e l e c t i v e reagent f o r sulfhydryl groups (47). Figure 15 shows the influence of divalent cations on the r e a c t i v i t y of membrane sulfhydryl groups towards DTNB, with r e s u l t s being expressed as increases or decreases i n DTNB incorpora-tion i n the presence of divalent cations r e l a t i v e to the control (in the absence of any added cations). A l l the 2+ 2 + cations, with the exception of Ni and Co , produced s i m i l a r increases i n DTNB incorporation, probably due to 2 + some r e l a t i v e l y non-specific e f f e c t . Ni caused a con-siderably larger increase i n DTNB incorporation than the 2 + other ions, while Co was the only cation i n the group 2 + which decreased DTNB incorporation. This e f f e c t with Co was probably due to complexation of sulfhydryl groups by the ion, thus e f f e c t i v e l y making them unavailable for reaction 55a F i g u r e 15 M o d i f i c a t i o n o f E r y t h r o c y t e Membrane S u l f h y d r y l G r oups by DTNB i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s Membranes were r e a c t e d w i t h DTNB (10 mM f i n a l c o n c e n t r a t i o n ) i n t h e p r e s e n c e o f v a r i o u s d i v a l e n t c a t i o n s (1 mM f i n a l c o n c e n t r a t i o n ) as d e s c r i b e d i n t h e Methods s e c t i o n , c e n t r i f u g e d , and t h e a b s o r b a n c e o f t h e s u p e r n a t a n t a t 412 nm u s e d t o c a l c u l a t e t h e number o f membrane s u l f h y d r y l g r o u p s m o d i f i e d . R e s u l t s a r e e x p r e s s e d as i n c r e m e n t a l i n c r e a s e s o r d e c r e a s e s i n DTNB i n c o r -p o r a t i o n r e l a t i v e t o t h e c o n t r o l i n t h e a b s e n c e o f added c a t i o n s . E a c h p o i n t r e p r e s e n t s t h e mean - s t a n d a r d e r r o r o f t h e mean o f e x p e r i m e n t s w i t h t h r e e d i f f e r e n t membrane p r e p a r a t i o n s . FIGURE 15 i s \ § a e x ^ ^ o ^ ^ Q o r y i BNIQ d I O ± r y ^ ! l A S 3 U 3 l \ 9 S w i t h DTNB. I t i s c l e a r , t h e r e f o r e , t h a t i n a d d i t i o n t o t h e 2+ 2 + q u a n t i t a t i v e d i f f e r e n c e s i n t h e a b i l i t y o f N i , Co , and 2 + Mn t o p e r t u r b membrane p r o t e i n s i t e s , as shown i n t h e TNBS l a b e l l i n g e x p e r i m e n t s , q u a l i t a t i v e d i f f e r e n c e s i n t h e s t r u c -t u r a l c o n s e q u e n c e s o f c a t ion-membrane i n t e r a c t i o n a l s o a r e a p p a r e n t f r o m t h e f o r e g o i n g e x p e r i m e n t s w i t h DTNB. As an e x t e n s i o n o f t h e a n a l y s i s o f i o n - i n d u c e d p r o t e i n s t r u c t u r a l a l t e r a t i o n s and an a p p r o a c h t o t h e p o s s i b l e f u n c t i o n a l c o n s e q u e n c e s o f c a t i o n - m e m b r a n e i n t e r a c t i o n , t h e i n f l u e n c e o f d i v a l e n t c a t i o n s on t h e f u n c t i o n i n g o f membrane-a s s o c i a t e d enzymes was s t u d i e d . I t was f e l t t h a t t h e s e ex-p e r i m e n t s m i g h t a l s o p r o v i d e i n f o r m a t i o n on t h e v e c t o r i a l c h a r a c t e r i s t i c s o f membrane-ion i n t e r a c t i o n s i n c e t h e e n -zymes c h o s e n f o r s t u d y d i f f e r e d i n t h e i r l o c a l i z a t i o n w i t h i n t h e membrane. A c e t y l c h o l i n e s t e r a s e i s an enzyme p r e s e n t a t t h e e x t e r n a l s u r f a c e o f t h e e r y t h r o c y t e membrane ( 5 0 ) , and i t m i g h t be e x p e c t e d t h a t any c o n f o r m a t i o n a l c h a n g e s i n d u c e d by i o n s a t t h e e x t e r i o r a s p e c t o f t h e membrane c o u l d a f f e c t t h e a c t i v i t y o f t h e enzyme. The r e s u l t s o f t h e e x p e r i m e n t s w i t h a c e t y l c h o l i n e s t e r a s e a r e p r e s e n t e d i n F i g u r e 16. I g -2+ n o r i n g Co f o r t h e moment, t h e e f f e c t s o f t h e o t h e r d i v a l e n t c a t i o n s , w h i l e f a i r l y s m a l l , were s u c h t h a t N i 2 + e x h i b i t e d a d i f f e r e n t p a t t e r n f r o m t h a t o f t h e o t h e r i o n s , w i t h t h e l a t t e r g i v i n g r i s e t o v a r y i n g d e g r e e s o f s t i m u l a t i o n b u t 2 + N i p r o d u c i n g i n h i b i t i o n o f t h e enzyme. T h i s o b s e r v a t i o n may be a f u r t h e r r e f l e c t i o n o f t h e d i s t i n c t i v e p r o p e r t i e s 2 + o f N i n o t e d e a r l i e r ( F i g u r e s 10 and 1 5 ) . The a p p a r e n t l y 5 7a F i g u r e 16 E f f e c t s o f D i v a l e n t C a t i o n s o n A c e t y l c h o l i n -e s t e r a s e A c t i v i t y A c e t y l c h o l i n e s t e r a s e a c t i v i t y o f e r y t h r o c y t e g h o s t s was d e t e r m i n e d i n t h e p r e s e n c e o f d i v a l e n t c a t i o n s (1 mM f i n a l c o n c e n t r a t i o n ) as d e s c r i b e d i n t h e Methods s e c t i o n . R e s u l t s a r e e x p r e s s e d i n t e r m s o f t h e d e g r e e o f i n h i b i t i o n r e l a t i v e t o t h e c o n t r o l i n t h e a b s e n c e o f a d d e d c a t i o n s . E a c h p o i n t r e p r e s e n t s t h e mean - s t a n d a r d e r r o r o f t h e mean o f e x p e r i m e n t s w i t h t h r e e d i f f e r e n t membrane p r e p a r a t i o n s . 57b FIGURE 16 58 l a r g e i n h i b i t i o n o f a c t i v i t y by C o i T i s more than l i k e l y due n o t to a major c o n f o r m a t i o n a l change a t the membrane s u r f a c e , but r a t h e r i n t e r f e r e n c e w i t h the assay o f t h i o c h o -l i n e l i b e r a t e d from the s u b s t r a t e a c e t y l t h i o c h o l i n e and e s t i m a t e d c o l o r i m e t r i c a l l y u s i n g DTNB. C o m p l e x a t i o n o f 2 + t h i o c h o l i n e s u l f h y d r y l groups by Co would i n t e r f e r e w i t h the DTNB r e a c t i o n and r e s u l t i n a low e s t i m a t e o f the number of s u b s t r a t e m o l e c u l e s a c t u a l l y degraded. Another enzyme used t o s t u d y the s t r u c t u r a l a l t e r a -t i o n s produced by d i v a l e n t c a t i o n s was membrane p - n i t r o -2 + phe n y l p h o s p h a t a s e (NPPase). T h i s enzyme r e q u i r e s Mg f o r a c t i v i t y and the o u a b a i n - s e n s i t i v e s t i m u l a t i o n o f the en-zyme by K + i s b e l i e v e d to r e f l e c t the t e r m i n a l dephos-p h o r y l a t i o n s t e p o f the N a + - and K + - s t i m u l a t e d ATPase system i n the membrane (51). The v e c t o r i a l c h a r a c t e r i s t i c s o f c a t i o n s t i m u l a t i o n a r e such t h a t t h e d i v a l e n t i o n i n t e r -a c t s w i t h the i n n e r s u r f a c e o f tne membrane w h i l e K + s t i m u -l a t i o n o c c u r s a t the o u t e r membrane s u r f a c e ( 5 2 ) . I t was hoped, i n v i e w of t h i s d i s t r i b u t i o n o f i o n - b i n d i n g s i t e s on o p p o s i t e s i d e s o f t h e membrane, t h a t a s t u d y o f d i v a l e n t c a t i o n e f f e c t s on the a c t i v i t y o f t h i s enzyme system might y i e l d d e t a i l e d i n f o r m a t i o n about the v e c t o r i a l c h a r a c t e r i s -t i c s o f c a t i o n - i n d u c e d membrane s t r u c t u r a l a l t e r a t i o n s . Two d i f f e r e n t t y p e s o f experiments were c a r r i e d o u t . F i r s t , 2 + Mg was s u b s t i t u t e d by the v a r i o u s d i v a l e n t c a t i o n s and b o t h b a s a l as w e l l as K + - s t i m u l a t e d a c t i v i t i e s were assayed. R e s u l t s shown i n F i g u r e 17 demonstrate the h i g h degree o f 59a F i g u r e 17 E f f e c t s o f S u b s t i t u t i n g D i v a l e n t C a t i o n s f o r 2+ + Mg on the A c t i v i t y o f B a s a l and K - St i m u -l a t e d p - N i t r o p h e n y l p h o s p h a t a s e B a s a l and K +- s t i m u l a t e d p - n i t r o p h e n y l p h o s p h a t a s e a c t i v i t i e s o f e r y t h r o c y t e g h o s t s were d e t e r m i n e d as d e s c r i b e d i n the Methods s e c t i o n , w i t h the 2 + Mg n o r m a l l y used i n the assay s u b s t i t u t e d by v a r i o u s d i v a l e n t c a t i o n s (1 mM f i n a l c o n c e n t r a t i o n ) . A c t i v i t y i s e x p r e s s e d i n terms o f nmoles p - n i t r o p h e n o l produced/hour/mg p r o t e i n . Each p o i n t r e p r e s e n t s the mean - s t a n d a r d e r r o r of the mean of e x p e r i m e n t s w i t h t h r e e d i f f e r e n t membrane p r e p a r a t i o n s . 59b FIGURE 17 .6 . a I . O 1.2 C R Y S T A L R A D I U S ( A ) s p e c i f i c i t y i n t h e d i v a l e n t c a t i o n r e q u i r e m e n t o f NPPase, 2+ 2 + s u c h t h a t o n l y Mg and Mn were a b l e t o s u p p o r t b a s a l a c t i v i t y and i t s s t i m u l a t i o n by K +. The l a c k o f e f f e c t i v e -n e s s o f t h e o t h e r d i v a l e n t c a t i o n s c o u l d e i t h e r r e s u l t f r o m t h e i r i n a b i l i t y t o i n t e r a c t w i t h t h e c r i t i c a l s i t e on t h e i n n e r membrane s u r f a c e o r f r o m t h e i r i n a b i l i t y t o i n d u c e a c a t a l y t i c a l l y a c t i v e c o n f o r m a t i o n i n t h e enzyme on c o m b i n a -t i o n w i t h t h i s s i t e . E v i d e n c e c o n s i s t e n t w i t h t h e l a t t e r p o s s i b i l i t y was o b t a i n e d f r o m e x p e r i m e n t s w h e r e i n t h e 2 + e f f e c t s o f v a r i o u s d i v a l e n t c a t i o n s on Mg - s t i m u l a t e d a c t i v i t y were d e t e r m i n e d ( F i g u r e 1 8 ) . A l l t h e i o n s e x c e p t 2 + Mn c a u s e d some d e g r e e o f i n h i b i t i o n o f b a s a l a c t i v i t y i n t h e a b s e n c e o f K +, p o s s i b l y as t h e r e s u l t o f c o m p e t i t i o n 2 + w i t h Mg a t i t s s i t e o f a c t i v a t i o n . R e g a r d l e s s o f t h e 2+ mechanism i n v o l v e d , however, t h e i m p a i r m e n t o f Mg -dep e n d -e n t NPPase a c t i v i t y d e m o n s t r a t e d t h a t m e m b r a n e - d i v a l e n t c a t i o n i n t e r a c t i o n p e r t u r b s t h e i n n e r membrane s u r f a c e f r o m w h i c h t h i s a c t i v i t y o r i g i n a t e s . I n a d d i t i o n , t h e g r e a t e r i n h i b i t i o n o f t h e K + - s t i m u l a t e d component r e l a t i v e t o t h e b a s a l a c t i v i t y may be t a k e n as e v i d e n c e t h a t c a t i o n s a r e a l s o a b l e t o c a u s e f u n c t i o n a l l y s i g n i f i c a n t s t r u c t u r a l a l t e r a t i o n s a t t h e o u t e r membrane s u r f a c e where K + e x e r t s i t s s t i m u l a t o r y a c t i o n . The p r e s e n t d a t a i n d i c a t i n g t h a t d i v a l e n t c a t i on-membrane i n t e r a c t i o n l e a d s to p e r t u r b a t i o n o f b o t h i n n e r and o u t e r membrane s u r f a c e s do n o t , however, a l l o w any c o n c l u s i o n s as t o t h e l o c a t i o n o f t h e c a t i o n b i n d i n g s i t e s m e d i a t i n g t h e s e e f f e c t s . 61a F i g u r e 18 E f f e c t s o f D i v a l e n t C a t i o n s on B a s a l and K + -S t i m u l a t e d p - N i t r o p h e n y l p h o s p h a t a s e A c t i v i t i e s B a s a l a n d K +- s t i m u l a t e d p - n i t r o p h e n y l p h o s p h a t a s e a c t i v i t i e s o f e r y t h r o c y t e g h o s t s w e re d e t e r m i n e d a s d e s c r i b e d i n t h e M e t h o d s s e c t i o n , w i t h d i v a l e n t c a t i o n s p r e s e n t a t a f i n a l c o n c e n t r a t i o n o f 1 mM i n a d d i t i o n t o t h e u s u a l c o n c e n t r a t i o n o f 2+ Mg . R e s u l t s a r e e x p r e s s e d as p e r c e n t i n h i b i -t i o n r e l a t i v e t o t h e c o n t r o l v a l u e w i t h no a d d i -t i o n a l d i v a l e n t c a t i o n s p r e s e n t . E a c h p o i n t r e p r e s e n t s t h e mean - s t a n d a r d e r r o r o f t h e mean o f e x p e r i m e n t s w i t h t h r e e d i f f e r e n t membrane p r e p a r a t i o n s . 61b FIGURE 18 C R Y S T A L R A D I U S ( A ) EFFECTS OF CATIONS ON HEMOLYSIS OF INTACT ERYTHROCYTES A l l the e x p e r i m e n t a l d a t a p r e s e n t e d thus f a r were ob-t a i n e d u s i n g h e m o g l o b i n - f r e e e r y t h r o c y t e membranes. I t was o f c o n s i d e r a b l e i n t e r e s t t o examine the i n f l u e n c e o f i n -o r g a n i c c a t i o n s , a t c o n c e n t r a t i o n s employed i n the f o r e -g o i n g l a b e l l i n g and enzyme s t u d i e s , on the p r o p e r t i e s o f i n t a c t e r y t h r o c y t e s . A c o n v e n i e n t e x p e r i m e n t a l model i s the h e m o l y t i c b e h a v i o u r o f human e r y t h r o c y t e s s u b j e c t e d t o h y p o t o n i c s t r e s s . R e s u l t s o f s t u d i e s u t i l i z i n g 1 mM con-c e n t r a t i o n s o f d i v a l e n t c a t i o n s and 10 mM c o n c e n t r a t i o n s o f monovalent c a t i o n s a r e p r e s e n t e d i n F i g u r e 19. The mono-v a l e n t c a t i o n s produced a s i z e - i n d e p e n d e n t d e c r e a s e i n h e m o l y s i s , p r o b a b l y due to i n c r e a s e d i o n i c s t r e n g t h . ( C h o l i n e , which does n o t p e n e t r a t e the membrane because o f i t s s i z e and c h a r g e , produced the same degree of p r o t e c t i o n from h e m o l y s i s as the monovalent c a t i o n s a t a c o n c e n t r a t i o n o f 10 mM). The d i v a l e n t c a t i o n s , on the o t h e r hand, p r o -duced e f f e c t s which were dependent on the i o n i c r a d i u s . I n 2 + a l l c a s e s , w i t h the e x c e p t i o n o f N i , h e m o l y s i s was de-c r e a s e d from the c o n t r o l v a l u e . I t may be n o t e d t h a t the h e m o l y s i s - i o n i c r a d i u s p r o f i l e s o f b o t h d i v a l e n t and mono-v a l e n t c a t i o n s b e a r a s t r o n g resemblance to p r e v i o u s p l o t s o f a c t i v a t i o n energy o f TNBS i n c o r p o r a t i o n and maximal TNBS i n c o r p o r a t i o n i n t o p r o t e i n . The d e v i a t i o n o f C o 2 + from the u s u a l p a t t e r n i n the h e m o l y s i s c u r v e may be due to i n c r e a s e d s t a b i l i z a t i o n o f membrane s u l f h y d r y l s w h i c h are known to be i m p o r t a n t i n d e t e r m i n i n g h e m o l y s i s (5 3 ) . With the e x c e p t i o n o f t h i s i o n , 'a c o r r e l a t i o n appeared to e x i s t ( r = 0.91) 63a F i g u r e 19 E f f e c t s o f M o n o v a l e n t and D i v a l e n t C a t i o n s on H y p o t o n i c H e m o l y s i s o f I n t a c t E r y t h r o c y t e s H e m o l y s i s o f i n t a c t e r y t h r o c y t e s was i n d u c e d i n t h e p r e s e n c e o f m o n o v a l e n t (10 mM t f i n a l c o n c e n -t r a t i o n ) and d i v a l e n t (1 mM f i n a l c o n c e n t r a t i o n ) c a t i o n s as d e s c r i b e d i n t h e Methods s e c t i o n . R e s u l t s a r e e x p r e s s e d as a p e r c e n t a g e o f t h e c o n t r o l h e m o l y s i s ( i n t h e a b s e n c e o f added c a t i o n ) , w h i c h a v e r a g e d 55-65% o f t h e t o t a l h e m o l y s i s ( i n d i s t i l l e d w a t e r ) . E a c h p o i n t r e p r e s e n t s t h e mean t s t a n d a r d e r r o r o f t h e mean o f t r i p l i c a t e e x p e r i m e n t s u s i n g t h r e e d i f f e r e n t b l o o d s a m p l e s . CTi tr between the p r o t e c t i o n from h e m o l y s i s by each d i v a l e n t c a t i o n i n i n t a c t e r y t n r o c y t e s ( F i g u r e 19) and the r a t i o o f p h o s p h o l i p i d t o p r o t e i n l a b e l l i n g by TNBS i n e r y t h r o c y t e membranes o f t h e s e same c a t i o n s ( T a b l e I ) . TURBIDITY MEASUREMENTS IN SONICATED MEMBRANE SUSPENSIONS In any s t u d i e s o f membrane s t r u c t u r a l c h a r a c t e r i s t i c s based on the i n c o r p o r a t i o n ( c o v a l e n t o r n o n - c o v a l e n t ) o f exogenous p r o b e s , whether they be chromophoric ( 3 7 , 3 8 ) , f l u o r e s c e n t ( 3 4 ) , o r s p i n l a b e l l e d ( 2 8 ) , t h e r e i s u s u a l l y some u n c e r t a i n t y as to the r o l e o f p e r t u r b a t i o n a l e f f e c t s i n d u c e d by the probe i t s e l f i n d e t e r m i n i n g the e x p e r i m e n t a l r e s u l t . I t was, t h e r e f o r e , c o n s i d e r e d d e s i r a b l e t o f i n d a means o f a n a l y z i n g d i v a l e n t c a t i o n - i n d u c e d membrane s t r u c -t u r a l a l t e r a t i o n s which would n o t i n v o l v e the use o f co-v a l e n t bond-forming c n e m i c a l p r o b e s . I t has r e c e n t l y been s u g g e s t e d t h a t e r y t h r o c y t e membrane t r a n s i t i o n t e mperatures may be d e t e r m i n e d by measuring the v i s c o s i t y o f s o n i c a t e d membrane s u s p e n s i o n s as a f u n c t i o n o f temperature (5 4 ) . The a u t h o r s c l a i m e d t h a t i f measured v i s c o s i t i e s were p l o t t e d a g a i n s t the i n v e r s e o f the a b s o l u t e t e m p e r a t u r e , one s h o u l d o b t a i n a s t r a i g h t - l i n e graph which e x h i b i t e d a break a t the t r a n s i t i o n t e mperature. S i n c e l i p i d s a r e t h e major d e t e r -minants o f such t h e r m a l t r a n s i t i o n s (55) and c a t i o n s have been shown he r e t o markedly p e r t u r b membrane p h o s p h o l i p i d s , i t was f e l t t h a t t h i s t e c h n i q u e might r e p r e s e n t a c o n v e n i e n t means o f f u r t h e r c h a r a c t e r i z i n g such ion-dependent s t r u c t u r a l a l t e r a t i o n s . U n f o r t u n a t e l y , when t h i s m ethod was t r i e d , i t was f o u n d t h a t t h e d a t a c o u l d b e s t be f i t t e d b y a c u r v e , r a t h e r t h a n two s t r a i g h t l i n e s , a n d t h e m e t h o d was, t h e r e -f o r e , n o t c o n s i d e r e d a c c e p t a b l e f o r d e t e r m i n g t r a n s i t i o n t e m p e r a t u r e s . I t was n o t e d , h o w e v e r , t h a t p r o n o u n c e d d i f f e r e n c e s i n t h e o p a c i t y o f membrane s u s p e n s i o n s s o n i c a t e d i n t h e p r e s e n c e o f c a t i o n s o c c u r r e d a n d t h i s was i n v e s t i g a t e d f u r t h e r . The r e s u l t s o f t h i s i n v e s t i g a t i o n a r e shown i n F i g u r e 20. A g a i n , t h e r e c u r r i n g p a t t e r n o f i o n i c r a d i u s p r o f i l e s o f b o t h d i v a l e n t a n d m o n o v a l e n t c a t i o n s may be o b -s e r v e d i n t h e s e e x p e r i m e n t s . The p o s s i b i l i t y t h a t t h e s e e f f e c t s may be due t o c a t i o n - i n d u c e d a g g r e g a t i o n o f membrane f r a g m e n t s p r o d u c e d b y s o n i c a t i o n was t e s t e d b y a d d i n g t h e c a t i o n s t o a membrane s u s p e n s i o n w h i c h h a d b e e n p r e v i o u s l y s o n i c a t e d . As c a n be s e e n f r o m F i g u r e 2 1 , a s i m i l a r i o n i c r a d i u s p r o f i l e i s o b t a i n e d w h e t h e r t h e i o n s a r e a d d e d b e f o r e o r a f t e r s o n i c a t i o n , b u t t h e i n c r e a s e i n a b s o r b a n c e o v e r t h e c o n t r o l v a l u e i s much more p r o n o u n c e d when t h e c a t i o n s a r e p r e s e n t d u r i n g s o n i c a t i o n , s u g g e s t i n g t h a t t h e o b s e r v e d r e -s u l t s a r e n o t due s o l e l y t o a g g r e g a t i o n o f membrane f r a g -m e n t s . The e f f e c t s o f p r i o r t r e a t m e n t o f t h e membranes w i t h n e u r a m i n i d a s e , t r y p s i n , and p h o s p h o l i p a s e A o n t h e a b s o r b -a n c e o f membrane s u s p e n s i o n s s o n i c a t e d i n t h e p r e s e n c e o f t h e v a r i o u s d i v a l e n t c a t i o n s was a l s o t e s t e d . F i g u r e 22 shows t h e r e s u l t s o f t h i s work. Membranes t r e a t e d w i t h n e u r a m i n i d a s e a n d s o n i c a t e d i n t h e p r e s e n c e o f t h e d i v a l e n t 66a F i g u r e 20 E f f e c t s o f M o n o v a l e n t a n d D i v a l e n t C a t i o n s o n U l t r a s o n i c D i s r u p t i o n o f E r y t h r o c y t e Membranes Membranes were s o n i c a t e d i n t h e p r e s e n c e o f m o n o v a l e n t (10 mM f i n a l c o n c e n t r a t i o n ) a n d d i v a l e n t (1 mM f i n a l c o n c e n t r a t i o n ) c a t i o n s as d e s c r i b e d i n t h e M e t h o d s s e c t i o n . The d e g r e e o f d i s r u p t i o n was e s t i m a t e d b y m e a s u r i n g t h e a b s o r b a n c e o f t h e r e s u l t i n g s u s p e n s i o n a t 900 nm. E a c h p o i n t r e p r e s e n t s t h e mean - s t a n d a r d e r r o r o f t h e mean o f t h r e e d i f f e r e n t membrane p r e p a r a t i o n s . 66b FIGURE 20 L U U O O B ± V 3'3 NVetiOSBV 67a F i g u r e 21 E f f e c t s o f D i v a l e n t C a t i o n s on the U l t r a s o n i c D i s r u p t i o n o f E r y t h r o c y t e Membranes Membranes were s o n i c a t e d i n the p r e s e n c e o f d i v a l e n t c a t i o n s (1 mM f i n a l c o n c e n t r a t i o n ) . A l t e r n a t i v e l y , membranes were s o n i c a t e d i n the absence o f c a t i o n s and c a t i o n s (1 mM f i n a l c o n -c e n t r a t i o n ) were added a f t e r the s o n i c a t i o n was completed. In b o t h c a s e s , the absorbance a t 900 nm o f the r e s u l t i n g s u s p e n s i o n was d e t e r -mined as d e s c r i b e d i n F i g u r e 20. FIGURE 21 . 6 " T .8 I.O i . a i.a C R Y S T A L R A D I U S ( A ) 68a F i g u r e 22 E f f e c t s o f D i v a l e n t C a t i o n s on t h e U l t r a s o n i c D i s r u p t i o n o f E n z y m a t i c a l l y M o d i f i e d E r y t h r o c y t e Membranes Membranes were p r e t r e a t e d w i t h t r y p s i n , n e u r a m i n i d a s e o r p h o s p h o l i p a s e A and t h e n s o n i c a t e d i n t h e p r e s e n c e o f d i v a l e n t c a t i o n s ( 1 mM f i n a l c o n c e n t r a t i o n ) as d e s c r i b e d i n t h e Methods s e c t i o n . The a b s o r b a n c e s o f t h e r e s u l t i n g s u s p e n s i o n s were d e t e r m i n e d a t 900 nm and compared t o t h e a b s o r b a n c e o f a c o n t r o l s a m p l e , s o n i c a t e d i n t h e a b s e n c e o f c a t i o n s . INSET: The i n s e t shows t h e a c t u a l a b s o r b a n c e r e a d i n g s a t 900 nm o f c o n t r o l membrane s a m p l e s , p r e t r e a t e d w i t h t h e v a r i o u s enzymes. FRACTION OF CONTROL ABSORBANCE zz a n r i D i J q89 2+ 2+ 2+ c a t i o n s o f N i , Co , and Mn d e m o n s t r a t e d a d e c r e a s e d a b s o r b a n c e r e l a t i v e t o t h e c o n t r o l (no i o n s p r e s e n t ) when c o m p a r e d t o membranes w h i c h h a d n o t b e e n t r e a t e d , w h i l e t h e e f f e c t s o f t h e a l k a l i n e e a r t h c a t i o n s o n s o n i c a t i o n a r e n o t c h a n g e d b y t h i s membrane t r e a t m e n t . I t i s i n t e r e s t i n g t o n o t e t h a t t h e v a r i a t i o n s among t h e i o n s f o u n d w i t h u n t r e a t e d membranes a r e c o m p l e t e l y a b o l i s h e d b y p r e t r e a t m e n t o f t h e membranes w i t h n e u r a m i n i d a s e , s u g g e s t i n g t h a t t h e s i a l i c a c i d c a r b o x y l s may be an i m p o r t a n t s i t e o f a c t i o n o f some 2+ 2+ 2 + o f t h e d i v a l e n t c a t i o n s , s p e c i f i c a l l y Co , N i , and Mn T h i s i s i n c o n t r a s t t o t h e i n t e r p r e t a t i o n o f e a r l i e r s t u d i e s o n e f f e c t s o f s i a l i c a c i d r e m o v a l o n TNBS i n c o r p o r a t i o n ( F i g u r e 9 ) , w h i c h i n d i c a t e d t h a t t h e s e a n i o n i c g r o u p s h a d no e f f e c t o n e i t h e r a c t i v a t i o n e n e r g y o r r a t e o f TNBS i n -2 + c o r p o r a t i o n . H owever, Mg was u s e d as a r e p r e s e n t a t i v e d i v a l e n t c a t i o n i n t h e s e s t u d i e s a n d t h e i n t e r p r e t a t i o n o f t h e r e s u l t s w o u l d be c o n s i s t e n t w i t h a l a c k o f e f f e c t o f n e u r a m i n i d a s e t r e a t m e n t o n e x p e r i m e n t s c o n d u c t e d i n t h e p r e s e n c e o f t h e a l k a l i n e e a r t h c a t i o n s . Membranes t r e a t e d w i t h t r y p s i n o r p h o s p h o l i p a s e A y i e l d e d c o n s i d e r a b l y d i f f e r e n t r e s u l t s t h a n t h o s e t r e a t e d w i t h n e u r a m i n i d a s e . N e i t h e r o f t n e s e t r e a t m e n t s r e s u l t e d i n t h e l o s s o f v a r i a t i o n among t h e i o n s , a n d t h e v a r i o u s c a t i o n s w e re a f f e c t e d d i f f e r e n t l y b y t h e s e two t r e a t m e n t s , s u g g e s t i n g d i f f e r e n t d e g r e e s o f d e p e n d e n c e on l i p i d a n d p r o -t e i n i n t e g r i t y f o r t h e i r e f f e c t s . T r y p s i n i z a t i o n o f mem-b r a n e s g e n e r a l l y d e c r e a s e d t h e a b s o r b a n c e i n t h e p r e s e n c e o f i o n s r e l a t i v e t o t h e c o n t r o l (no i o n s p r e s e n t ) when com-2 + p a r e d t o u n t r e a t e d membranes. Mn was an e x c e p t i o n t o t h i t r e n d , d e m o n s t r a t i n g an i n c r e a s e i n t h e r e l a t i v e a b s o r b a n c e 2 + I t may a l s o be n o t e d t n a t Co , a l t n o u g h s h o w i n g a d e c r e a s e i n r e l a t i v e a b s o r b a n c e , was n o t a f f e c t e d n e a r l y as much as t h e o t h e r c a t i o n s i n t h i s s t u d y . P h o s p h o l i p a s e A t r e a t m e n t a l s o r e s u l t e d i n a d e c r e a s e d r e l a t i v e a b s o r b a n c e f o r a l l t h e c a t i o n s , b u t a g a i n , t h e d e g r e e o f t h e e f f e c t v a r i e d 2 + somewhat. N i was l e a s t a f f e c t e d , w h i l e t h e o t h e r c a t i o n s d e m o n s t r a t e d a d e c r e a s e t o a p p r o x i m a t e l y o n e - h a l f o f t h e v a l u e f o u n d w i t h u n t r e a t e d membranes. R e g a r d l e s s o f t h e t y p e o f membrane p r e t r e a t m e n t , i t may be n o t e d t h a t t h e a l k a l i n e e a r t h c a t i o n s a l w a y s e x h i b i t s i m i l a r b e h a v i o u r as a c l a s s . T h i s i s c o n s i s t e n t w i t h e a r l i e r s t u d i e s on t h e e f f e c t s o f enzyme t r e a t m e n t on TNBS l a b e l l i n g ( F i g u r e 11) and w i t h some a s p e c t s o f TNBS i n c o r p o r a t i o n ( F i g u r e 10, u p p e r ) and col u m n work ( F i g u r e 13, l o w e r ) . ION-INDUCED PERTURBATIONS IN OTHER MEMBRANE SYSTEMS T h i s f i n a l s e c t i o n d e s c r i b e s p r e l i m i n a r y a t t e m p t s t o a p p l y some o f t h e t e c h n i q u e s d e v e l o p e d h e r e f o r the s t u d y o f e r y t h r o c y t e membranes t o o t h e r b i o l o g i c a l membranes. B o v i n e s y n a p t i c v e s i c l e membranes and r a t l i v e r m i c r o s o m a l membranes were c h o s e n f o r s t u d y as t y p i c a l membrane s y s t e m s f r o m e l e c t r i c a l l y e x c i t a b l e and n o n - e x c i t a b l e t i s s u e s r e -s p e c t i v e l y . The b o v i n e s y n a p t i c v e s i c l e s and p u r i f i e d r a t l i v e r m i c r o s o m a l membranes were l a b e l l e d w i t h TNBS and s o l u b i l i z e d i n t h e same manner as e r y t h r o c y t e membranes, and the l a b e l l e d p r o t e i n and p h o s p h o l i p i d components s e p a r a t e d by g e l f i l t r a t i o n on Sephadex G-200. As w i t h the e r y t h r o -c y t e membranes, the pr e s e n c e o f d i v a l e n t c a t i o n s caused an i n c r e a s e i n TNBS i n c o r p o r a t i o n i n t o b o t h the p r o t e i n and p h o s p h o l i p i d components. Maximal TNBS i n c o r p o r a t i o n i n t o p r o t e i n and p h o s p h o l i p i d o f b o v i n e s y n a p t i c v e s i c l e s and p u r i f i e d r a t l i v e r microsomal membranes (as d e t e r m i n e d by the absorbance a t 3 35 nm) i n the pr e s e n c e o f each c a t i o n i s shown as a f u n c t i o n o f i o n i c r a d i u s i n F i g u r e s 23 and 24. R e f e r e n c e t o F i g u r e 13 i n d i c a t e s some d i f f e r e n c e s i n the c a t i o n e f f e c t s as compared to e r y t h r o c y t e membranes. Wi t h the b o v i n e s y n a p t i c v e s i c l e s , i t may be n o t i c e d t h a t the maximum p r o t e i n l a b e l l i n g o c c u r s i n the pr e s e n c e o f 2+ 2 + Co , r a t h e r than N i as found w i t h e r y t h r o c y t e membranes The p h o s p h o l i p i d l a b e l l i n g o f th e s e v e s i c l e s shows no d i f f e r e n t i a t i o n among the a l k a l i n e e a r t h c a t i o n s , w h i l e i n e r y t h r o c y t e membranes, t h e r e a r e n o t i c e a b l e v a r i a t i o n s i n the e x t e n t o f p h o s p h o l i p i d l a b e l l i n g i n the pr e s e n c e o f 2+ 2+ 2+ 2 + Mg , Ca , Sr , and Ba . With the p u r i f i e d r a t l i v e r m i crosomal membranes, t h e maximal p r o t e i n l a b e l l i n g p r o -f i l e r esembles t h a t o f the e r y t h r o c y t e s , b u t the phos-p h o l i p i d p r o f i l e i s c o n s i d e r a b l y d i f f e r e n t , w i t h the maxi-2+ 2 + mum l a b e l l i n g o c c u r r i n g i n t h e pr e s e n c e o f N i and Mn 2 + r a t h e r than Co , and a f l a t t e n i n g o f the l a b e l l i n g p r o f i l as compared to e r y t h r o c y t e s b e i n g a p parent. R a t i o s o f maximal p h o s p h o l i p i d to maximal^ p r o t e i n l a b e l l i n g i n the presence o f c a t i o n s were c a l c u l a t e d f o r 72a F i g u r e 23 Maximal TNBS L a b e l l i n g o f P r o t e i n and Phospho-l i p i d Components o f Bovine S y n a p t i c V e s i c l e s i n t he Presence o f D i v a l e n t C a t i o n s Membranes were l a b e l l e d w i t h TNBS and t r e a t e d as d e s c r i b e d i n F i g u r e 12 e x c e p t t h a t 1.5 ml r a t h e r than 2.0 ml membrane s u s p e n s i o n were u t i l i z e d . Maximal absorbances o f the r e s o l v e d p r o t e i n and p h o s p h o l i p i d components a t 335 nm were then determined. 72b FIGURE 23 C R Y S T A L R A D I U S ( A ) 7 3a F i g u r e 24 Maximal TNBS L a b e l l i n g o f P r o t e i n and Phospho-l i p i d Components o f P u r i f i e d R a t L i v e r M i c r o s o m a l Membranes i n t h e P r e s e n c e o f D i v a l e n t C a t i o n s Membranes were l a b e l l e d w i t h TNBS and t r e a t e d as d e s c r i b e d i n F i g u r e 12 e x c e p t t h a t 1.5 ml r a t h e r t h a n 2.0 ml membrane s u s p e n s i o n were u t i l i z e d . Maximal a b s o r b a n c e s o f t h e r e s o l v e d p r o t e i n and p h o s p h o l i p i d components a t 335 nm were t h e n d e t e r m i n e d f o r e a c h c a t i o n . FIGURE 24 C R Y S T A L R A D I U S ( A ) b o t h t h e b o v i n e s y n a p t i c v e s i c l e s and p u r i f i e d r a t l i v e r m i c r o s o m a l membranes. R e s u l t s a r e summarized i n T a b l e I I . As w i t h e r y t h r o c y t e membranes, t h e c a t i o n s t e n d t o s e p a r a t e i n t o two g r o u p s , one c o n s i s t i n g o f t h e a l k a l i n e e a r t h 2 + c a t i o n s and Mn w i t h r a t i o s o f a b o u t 4 f o r b o v i n e s y n a p t i c v e s i c l e s and a b o u t 1.2 f o r p u r i f i e d r a t l i v e r m i c r o s o m a l 2+ 2 + membranes, t h e o t h e r g r o u p c o n s i s t i n g o f Co and N i w i t h r a t i o s o f a b o u t 2.5 and 0.9 f o r b o v i n e s y n a p t i c v e s i c l e s and p u r i f i e d r a t l i v e r m i c r o s o m a l membranes r e s p e c t i v e l y . I t may be n o t e d t h a t , u n l i k e t h e e r y t h r o c y t e membranes, t h e p h o s p h o l i p i d l a b e l l i n g i s l e s s t h a n t h e p r o t e i n l a b e l l i n g i n t h e a b s e n c e o f i o n s i n b o t h membrane p r e p a r a t i o n s . I n t h e p r e s e n c e o f c a t i o n s , t h e p h o s p h o l i p i d l a b e l l i n g i s s t i m u -l a t e d p r e f e r e n t i a l l y , b u t i n t h e p u r i f i e d r a t l i v e r m i c r o -2+ 2 + somal membranes i n t h e p r e s e n c e o f Co and N i , t h e p h o s -p h o l i p i d l a b e l l i n g s t i l l d oes n o t e x c e e d t h e p r o t e i n l a b e l l i n g . S o n i c a t i o n s t u d i e s i n t h e p r e s e n c e o f c a t i o n s were a l s o u n d e r t a k e n w i t h b o v i n e s y n a p t i c v e s i c l e s , r a t l i v e r m i c r o s o m e s , and p u r i f i e d r a t l i v e r m i c r o s o m a l membranes. R e s u l t s o f t h e s e e x p e r i m e n t s a r e shown i n F i g u r e 25. A g a i n , as w i t h e r y t h r o c y t e membranes ( F i g u r e 2 0 ) , t h e same i o n i c r a d i u s p r o f i l e s a r e o b t a i n e d w i t h a l l t h r e e membrane p r e -p a r a t i o n s , a l t h o u g h t h e r e l a t i v e e f f e c t i v e n e s s o f t h e c a t i o n s i n t h e d i f f e r e n t s y s t e m s v a r i e s . Table I I . I n f l u e n c e o f c a t i o n s on r e l a t i v e l a b e l l i n g o f b o v i n e s y n a p t i c v e s i c l e and p u r i f i e d r a t l i v e r m i crosomal membrane p h o s p h o l i p i d s and p r o t e i n s by TNBS c a t i o n * r a t i o maximal p h o s p h o l i p i d l a b e l l i n g maximal p r o t e i n l a b e l l i n g Bovine s y n a p t i c v e s i c l e s P u r i f i e d Rat L i v e r M i c r o s o m a l Membranes .none _Mg Ba 0.88 0.78 2 + 3.64 1.14 C a 2 + 4.35 1.19 S r 2 + 4.18 1.17 2 + 3.77 1.17 M n 2 + 4.03 1.10 , N i 2 + 2.78 0.89 C o 2 + 2.34 0.91 * D i v a l e n t c a t i o n s were t e s t e d a t a c o n c e n t r a t i o n o f 1 mM. Membranes were l a b e l l e d and chromatographed as d e s c r i b e d f o r F i g . 12, e x c e p t t h a t 1.5 ml were l a b e l l e d i n s t e a d o f 2.0 ml. Maximal p h o s p h o l i p i d and p r o t e i n l a b e l l i n g were e s t i m a t e d from the maximal absorbance a t 335 nm o f r e s o l v e d p h o s p h o l i p i d and p r o t e i n peaks f o r each i o n . 76a F i g u r e 25 E f f e c t s o f D i v a l e n t C a t i o n s on U l t r a s o n i c D i s -r u p t i o n o f Rat L i v e r M i c r o s o m e s , B o v i n e S y n a p t i c V e s i c l e s , and P u r i f i e d Rat L i v e r M i c r o s o m a l Membranes Membranes were s o n i c a t e d i n t h e p r e s e n c e o f d i v a l e n t c a t i o n s (1 mM f i n a l c o n c e n t r a t i o n ) as d e s c r i b e d i n t h e Methods s e c t i o n . The d e g r e e o f d i s r u p t i o n was e s t i m a t e d by m e a s u r i n g t h e a b s o r b a n c e s o f t h e r e s u l t i n g s u s p e n s i o n s a t 900 nm. (BSV = b o v i n e s y n a p t i c v e s i c l e s , ) PRLMM = p u r i f i e d r a t l i v e r m i c r o s o m a l membranes, RLM = r a t l i v e r m i c r o s o m e s ) . 76b FIGURE 25 C R Y S T A L R A D I U S ( A ) DISCUSSION As a r e s u l t o f t h e many s t u d i e s w h i c h h a v e e x p l o r e d t h e i n t e r a c t i o n o f d i v a l e n t c a t i o n s w i t h b i o l o g i c a l s y s t e m s , t h e c r u c i a l r o l e o f t h e s e i o n i c s p e c i e s i n g o v e r n i n g t h e f u n c t i o n a l p r o p e r t i e s o f l i v i n g t i s s u e s has become a p p a r e n t (1, 3, 4, 8, 14, 16, 1 7 ) . However, the p a u c i t y o f i n f o r m a -t i o n r e g a r d i n g t h e s t r u c t u r a l c o n s e q u e n c e s o f d i v a l e n t c a t i o n i n t e r a c t i o n has as y e t p r e c l u d e d a d e t a i l e d u n d e r s t a n d i n g o f t h e m o l e c u l a r a c t i o n s o f d i v a l e n t c a t i o n s a n d how t h e s e a c t i o n s r e l a t e t o f u n c t i o n a l c h a n g e s . The p r e c e e d i n g s t u d y a p p r o a c h e d t h i s p r o b l e m by a t t e m p t i n g t o l e a r n more a b o u t t h e m o l e c u l a r c o n s e q u e n c e s o f d i v a l e n t c a t i o n i n t e r a c t i o n w i t h b i o l o g i c a l membranes, f o c u s s i n g on t h e human e r y t h r o c y t e g h o s t as a model membrane s y s t e m . I t seems p r o b a b l e t h a t d i v a l e n t c a t i o n e f f e c t s on mem-b r a n e s a r e due to some c o m b i n a t i o n o f c h a r g e n e u t r a l i z a t i o n and c o n f i g u r a t i o n a l a l t e r a t i o n s , w i t h p e r t u r b a t i o n o f b o t h p r o t e i n and p h o s p h o l i p i d membrane components most l i k e l y b e i n g i n v o l v e d . The i n f l u e n c e o f d i v a l e n t c a t i o n s on s u r -f a c e p r e s s u r e and s u r f a c e p o t e n t i a l i n a r t i f i c i a l p h o s p h o -l i p i d s y s t e m s has i n d i c a t e d t h a t a c i d i c p h o s p h o l i p i d s i n t e r -a c t s t r o n g l y w i t h t h e s e i o n s ( 2 3 ) . T r a n s i t i o n t e m p e r a t u r e s o f p h o s p h o l i p i d s a r e a l s o a f f e c t e d by t h e p r e s e n c e o f d i v a l e n t c a t i o n s s u c h as C a ^ + , and v a r i a t i o n s i n t h e m o r p h o l o g y o f l i p s o m a l s t r u c t u r e s c a n be d e m o n s t r a t e d i n t h e p r e s e n c e o f t h e s e i o n s ( 3 3 ) . I n a d d i t i o n , c e r t a i n membrane-bound enzymes a r e c r i t i c a l l y d e p e n d e n t on d i v a l e n t c a t i o n s f o r a c t i v i t y . A d e n y l c y c l a s e , w h i c h c a t a l y z e s t h e f o r m a t i o n o f c y c l i c 2 + 3 1 , 5 1 - a d e n o s i n e monophosphate, r e q u i r e s Mg f o r p p t i m a l 2 + 2 + a c t i v i t y . Mn c a n r e p l a c e Mg c o m p l e t e l y i n p r e p a r a t i o n s o f t h i s enzyme f r o m b r a i n , and p a r t i a l l y i n h e a r t p r e p a r a -2+ 2 + t i o n s , w h i l e o t h e r d i v a l e n t c a t i o n s s u c h as Ca , Co , and 2+ 2 + Zn c a n n o t s u b s t i t u t e a p p r e c i a b l y f o r Mg ( 5 6 ) . The 2 + enzyme a d e n o s i n e t r i p h o s p h a t a s e (ATPase) a l s o r e q u i r e s Mg f o r a c t i v i t y . I n a p r e p a r a t i o n o f f r a g m e n t e d r a t l i v e r 2+ 2+ 2+ m i t o c h o n d r i a , Mg c o u l d be s u b s t i t u t e d by Mn , Co , and 2 + Fe w i t h v a r y i n g d e g r e e s o f e f f e c t i v e n e s s , w h i l e o t h e r 2+ 2+ 2 + c a t i o n s s u c h as Zn , Cu , and Ca d i d n o t a c t i v a t e t h e enzyme ( 5 7 ) . I n some i n s t a n c e s , d i v a l e n t c a t i o n s may i n t e r -a c t d i r e c t l y w i t h an enzyme s u b s t r a t e , t h e r e b y a l t e r i n g i t s a b i l i t y t o i n t e r a c t w i t h t h e enzyme, as a p p e a r s t o be t h e c a s e w i t h c e r t a i n A T P - d e p e n d e n t enzymes ( 5 8 ) . More g e n e r a l l y , however, t h e p r e s e n c e o f d i v a l e n t c a t i o n s f o r enzyme a c t i v i t y may be n e c e s s a r y t o d i r e c t l y c o n v e r t enzyme c o n f i g u r a t i o n s t o c a t a l y t i c a l l y a c t i v e forms a n d / o r a t t a i n t h i s e f f e c t i n -d i r e c t l y by a l t e r i n g t h e s u r r o u n d i n g l i p i d s , s i n c e b o t h o f t h e above m e n t i o n e d enzymes a r e c r i t i c a l l y d e p e n d e n t on p h o s -p h o l i p i d s f o r a c t i v i t y ( 5 9 ) . Thus i t i s l i k e l y t h a t b i o -l o g i c a l l y i m p o r t a n t e f f e c t s o f i o n s may o f t e n i n v o l v e b o t h l i p i d and p r o t e i n m o i e t i e s . I n t h e p r e s e n t s t u d i e s , b o t h d i v a l e n t and m o n o v a l e n t c a t i o n s were f o u n d t o s t i m u l a t e t t r i n i t r o b e n z e n s u l f o n i c a c i d (TNBS) i n c o r p o r a t i o n i n t o mem-b r a n e amino g r o u p s ( F i g u r e s 6 and 7 ) , a l t h o u g h marked d i f f e r e n c e s i n t h e s i z e d e p e n d e n c e and t h e m a g n i t u d e o f t h e s e f f e c t s e x i s t e d between m o n o v a l e n t and d i v a l e n t c a t i o n s . S i n c e model s t u d i e s w i t h b u t y l a m i n e i n d i c a t e d t h a t n e i t h e r d i v a l e n t n o r m o n o v a l e n t c a t i o n s s i g n i f i c a n t l y a f f e c t e d t h e i n t r i n s i c r e a c t i v i t y o f amino g r o u p s t o w a r d TNBS, i t was c o n e l u d e d t h a t t h e d i v a l e n t c a t i o n s t i m u l a t i o n o f TNBS i n c o r -p o r a t i o n was t h e r e s u l t o f membrane p e r t u r b a t i o n s a f f e c t i n g membrane amino g r o u p r e a c t i v i t y i n d i r e c t l y , w h i l e m o n o v a l e n t c a t i o n s t i m u l a t i o n o f TNBS i n c o r p o r a t i o n was most l i k e l y due t o a n o n - s p e c i f i c i o n i c s t r e n g t h e f f e c t . I t may be n o t e d f r o m F i g u r e 13 t h a t t h e d i v a l e n t i o n s d i f f e r e d i n t h e i r a b i l i t y t o a f f e c t p r o t e i n o r p h o s p h o l i p i d membrane components. The maximal TNBS i n c o r p o r a t i o n i n t o 2 + p r o t e i n o c c u r r e d i n t h e p r e s e n c e o f N i w h i l e t h e maximal 2 + TNBS i n c o r p o r a t i o n i n t o p h o s p h o l i p i d o c c u r r e d w i t h Co R e f e r e n c e t o F i g u r e 6 i n d i c a t e s t h a t t h e p r o t e i n l a b e l l i n g t e n d e d t o p a r a l l e l t h e a c t i v a t i o n e n e r g y p r o f i l e w h i l e t h e p h o s p h o l i p i d l a b e l l i n g t e n d e d t o p a r a l l e l t h e r a t e o f TNBS i n c o r p o r a t i o n . T h e s e f i n d i n g s s u g g e s t e d t h a t membrane p r o -t e i n s may be i m p o r t a n t i n d e t e r m i n i n g t h e a c t i v a t i o n e n e r g y p a r a m e t e r and membrane p h o s p h o l i p i d s t h e r a t e p a r a m e t e r . I n a t t e m p t i n g t o f i n d some p h y s i c o - c h e m i c a l b a s i s f o r i n t e r p r e t i n g t h e above o b s e r v a t i o n s , i t was n o t e d t h a t a q u a l i t a t i v e c o r r e l a t i o n a p p e a r e d t o e x i s t between t h e f i r s t and s e c o n d i o n i z a t i o n p o t e n t i a l s o f d i v a l e n t c a t i o n s (49) and d i v a l e n t c a t i o n e f f e c t s on t h e a c t i v a t i o n e n e r g y and r a t e , r e s p e c t i v e l y , o f TNBS i n c o r p o r a t i o n ( F i g u r e s 6 and 8 ) . Th i s s u g g e s t e d t h a t the p r o p e r t i e s o f d i v a l e n t c a t i o n s t h a t d e t e r m i n e t h e i r i o n i z a t i o n p o t e n t i a l s may a l s o be r e l e v a n t t o membrane-cation i n t e r a c t i o n s . The f i r s t i o n i z a t i o n po-t e n t i a l s a r e dependent p r i m a r i l y on c a t i o n s i z e ( w i t h the 2 + e x c e p t i o n o f Mg , which i s anomalous w i t h r e s p e c t t o many p r o p e r t i e s (36, 4 9 ) ) , and they t e n d to p a r a l l e l the a c t i v a -t i o n energy p r o f i l e . Thus i t i s p o s s i b l e t h a t the e x p e r i -m e n t a l l y d e t e r m i n e d a c t i v a t i o n energy f o r each i o n i s de-pendent t o a l a r g e e x t e n t on c a t i o n s i z e . The s i m p l e s t mechanism t h a t would a c c o u n t f o r a dependence a l m o s t ex-c l u s i v e l y on c a t i o n s i z e i s t h a t of a masking o f n e g a t i v e charges on the membrane by s c r e e n i n g o r b i n d i n g o r a com-b i n a t i o n o f b o t h , s u c h t h a t t h e n e g a t i v e s u r f a c e c h a r g e , w h i c h tends to r e p e l the a n i o n i c TNBS m o l e c u l e , i s reduced. T h i s i s c o n s i s t e n t w i t h r e s u l t s o f o t h e r s t u d i e s on d i v a l e n t c a t i o n e f f e c t s w h i c h a l s o s u g g e s t charge n e u t r a l i z a t i o n as a p o s s i b l e mechanism of a c t i o n (3, 19, 31, 34, 35, 36). F u r t h e r s u p p o r t f o r t h i s p r o p o s a l t h a t the a c t i v a t i o n energy i s dependent on e l e c t r o s t a t i c p r o p e r t i e s a f f e c t i n g probe p e r m e a t i o n can be found i n the s t u d i e s o f the i n c o r p o r a t i o n o f p i c r y l c h l o r i d e (PC), a n e u t r a l t r i n i t r o p h e n y l a t i n g analogue o f TNBS, i n t o membranes. I n c o n t r a s t to t h e s i t u a -t i o n w i t h the a n i o n i c TNBS m o l e c u l e , d i v a l e n t c a t i o n s ( w i t h 2 + the e x c e p t i o n of N i ) d i d n o t a p p r e c i a b l y a l t e r t he a c t i v a -t i o n energy f o r PC i n c o r p o r a t i o n ( F i g u r e 1 0 ) , as would be ex p e c t e d f o r the p e r m e a t i o n o f an uncharged m o l e c u l e where the e f f e c t s o f charge n e u t r a l i z a t i o n s h o u l d be m i n i m a l . A l s o , the h i g h e r a c t i v a t i o n energy f o r u n s t i m u l a t e d i n c o r -p o r a t i o n o f TNBS (7.26 k c a l / m o l e ) as compared to PC (6.26 k c a l / m o l e ) i s c o n s i s t e n t w i t h the p r e s e n c e o f an e l e c t r o -s t a t i c b a r r i e r to TNBS p e r m e a t i o n . The second i o n i z a t i o n p o t e n t i a l s were found t o p a r a l l e l the r a t e s o f TNBS i n c o r p o r a t i o n ( F i g u r e s 6 and 8) and the p h o s p h o l i p i d l a b e l l i n g p a t t e r n ( F i g u r e 1 3 ) . I t has been e s t a b l i s h e d t h a t the p o l a r head groups o f membrane phospho-l i p i d s a r e n o r m a l l y a s s o c i a t e d w i t h an aqueous h y d r a t i o n s h e l l (60, 6 1 ) , and t h a t t h i s p o l a r head group h y d r a t i o n i s i m p o r t a n t i n d e t e r m i n i n g the s t r u c t u r a l o r g a n i z a t i o n o f the l i p i d s (62). D i v a l e n t c a t i o n s can compete f o r t h e water b i n d i n g s i t e s o f p h o s p h o l i p i d s , l e a d i n g t o d e h y d r a t i o n (63, 64) and consequent a l t e r a t i o n s i n c o n f i g u r a t i o n o f the p h o s p h o l i p i d m o i t i e s . I f a mechanism o f t h i s t y pe i s op-e r a t i o n a l h e r e , i t might be e x p e c t e d t o q u a l i t a t i v e l y r e -semble the second i o n i z a t i o n p o t e n t i a l , e s p e c i a l l y i f p a r t i a l removal o f c o o r d i n a t e d water from the d i v a l e n t c a t i o n s i s f i r s t n e c e s s a r y b e f o r e the i o n s can i n t e r a c t w i t h the p h o s p h o l i p i d s . By t h i s mechanism, b o t h the c a t i o n and p h o s p h o l i p i d would l o s e some o f t h e i r r e s p e c t i v e water mole-c u l e s o f h y d r a t i o n and r e p l a c e the water a s s o c i a t i o n w i t h a c a t i o n - p h o s p h o l i p i d i n t e r a c t i o n . T h i s v a r i a t i o n i n bound water i n the p r e s e n c e o f c a t i o n s may t r i g g e r l i p i d c o n-f i g u r a t i o n a l a l t e r a t i o n s w h i c h , i n t u r n , i n c r e a s e the r a t e o f TNBS i n c o r p o r a t i o n by e x p o s i n g p r e v i o u s l y i n a c c e s s i b l e amino groups./ I t has a l s o been proposed t h a t d i v a l e n t c a t i o n - i n d u c e d changes i n p h o s p h o l i p i d h y d r a t i o n and the r e s u l t a n t c o n f i g u r a t i o n a l changes may be r e s p o n s i b l e f o r changes i n membrane p e r m e a b i l i t y (65 ) y w h i c h , i n t u r n , i s i m p l i c a t e d i n t h e g e n e r a t i o n o f a c t i o n p o t e n t i a l s i n ex-c i t a b l e c e l l s ( 6 ) . The approaches d e v e l o p e d i n the s e s t u d i e s may p r o v i d e a means f o r e x p l o r i n g the r o l e of d i v a l e n t c a t i o n s i n d e t e r m i n i n g the e l e c t r i c a l p r o p e r t i e s o f e x c i t a b l e t i s s u e s . P r e l i m i n a r y e x p e r i m e n t s a l o n g t h e s e l i n e s a r e de-s c r i b e d l a t e r i n t h i s s e c t i o n . S i n c e , as n o t e d e a r l i e r , a c t i v a t i o n energy and p r o t e i n l a b e l l i n g p r o f i l e s f o r d i v a l e n t c a t i o n s ( F i g u r e s 6 and 13) were r a t h e r a n a l o g o u s , enzyme p r e t r e a t m e n t s o f membranes were u n d e r t a k e n i n o r d e r t o d e t e r m i n e whether o r not a r e l a t i o n s h i p a c t u a l l y e x i s t e d between p r o t e i n s and a c t i v a t i o n energy. The r e s u l t s o b t a i n e d i n d i c a t e d t h a t i t was h i g h l y l i k e l y t h a t p r o t e i n s p l a y an i m p o r t a n t r o l e i n the d e t e r -m i n a t i o n of a c t i v a t i o n e n e r g i e s . P h o s p h o l i p a s e A, which c o n v e r t s the membrane p h o s p h o l i p i d s t o c o r r e s p o n d i n g l y s o d e r i v a t i v e s by the h y d r o l y s i s o f y8 - a c y l l i n k a g e s ( 6 6 ) , t h e r e b y markedly a l t e r i n g membrane f l u i d i t y ( 6 7 ) , has l i t t l e e f f e c t on a c t i v a t i o n e n e r g i e s o f most o f t h e d i v a l e n t c a t i o n s , w h i l e t r y p s i n i z a t i o n , which c l e a v e s p r o t e i n bonds i n v o l v i n g a r g i n i n e o r l y s i n e r e s i d u e s ( 6 8 ) , d e c r e a s e s the a b i l i t y o f d i v a l e n t c a t i o n s t o l o w e r a c t i v a t i o n e n e r g i e s 2+ 2 + ( F i g u r e 11, u p p e r ) . The a c t i o n s o f N i and Co may be d i s t i n g u i s h e d from t h o s e o f the o t h e r d i v a l e n t c a t i o n s b o t h by t h e i r i n s e n s i t i v i t y t o t r y p s i n i z a t i o n e f f e c t s on a c t i v a -t i o n energy, and by t h e d i f f e r e n c e s n o t e d i n t h e i r e f f e c t s on t h e i n c o r p o r a t i o n o f 5 , 5 ' - d i t h i o - b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB) i n t o membrane p r o t e i n s u l f h y d r y l groups ( F i g u r e 1 5 ) . The anomalously h i g h a c t i v a t i o n energy o f PC i n c o r p o r a t i o n 2+ i n t h e p r e s e n c e o f N i i s a n o t h e r i n d i c a t i o n o f i t s u n i q u e 2 + membrane a c t i o n s , s u g g e s t i n g t h a t N i may a f f e c t probe p e r m e a t i o n i n a manner not e n t i r e l y r e l a t e d t o e l e c t r o s t a t i c c h a r a c t e r i s t i c s . I n c o n j u n c t i o n w i t h t h e s e s t u d i e s i n d i c a t i n g the im-p o r t a n c e of p r o t e i n s i n d e t e r m i n i n g a c t i v a t i o n e n e r g i e s , a s e r i e s o f e x p e r i m e n t s was a l s o conducted on n e u r a m i n i d a s e -t r e a t e d membranes. I f , as seems l i k e l y , e l e c t r o s t a t i c p r o -p e r t i e s a f f e c t i n g probe p e r m e a t i o n a r e i m p o r t a n t i n d e t e r -m i n i n g a c t i v a t i o n e n e r g i e s , n e u r a m i n i d a s e - s e n s i t i v e membrane s i a l i c a c i d g roups, which a r e r e s p o n s i b l e f o r most o f the n e g a t i v e charge a t the e x t e r n a l s u r f a c e o f the membrane ( 6 9 ) , might r e p r e s e n t a p o t e n t i a l s i t e o f d i v a l e n t c a t i o n a c t i o n . S i n c e t r y p s i n i z a t i o n can cause r e l e a s e o f membrane s i a l i c a c i d ( 7 0 ) , as w e l l as a f f e c t i n g p r o t e i n s , i t seemed p o s s i b l e t h a t the changes i n a c t i v a t i o n energy seen w i t h t r y p s i n i z e d membranes were due t o changes i n s i a l i c a c i d r a t h e r t h a n t o a l t e r a t i o n s i n p r o t e i n . However, n e u r a m i n i d a s e - t r e a t e d mem-branes e x h i b i t e d no d i f f e r e n c e s i n a c t i v a t i o n e n e r g i e s o r r a t e s o f e i t h e r b a s a l o r d i v a l e n t c a t i o n - s t i m u l a t e d TNBS i n -c o r p o r a t i o n ( F i g u r e 9 ) , i n d i c a t i n g t h a t s i a l i c a c i d groups ar e n o t i m p o r t a n t i n d e t e r m i n i n g a c t i v a t i o n energy. The same s e r i e s of e x p e r i m e n t s on e n z y m a t i c a l l y -m o d i f i e d membranes i n d i c a t e d t h e dependence o f the r a t e o f TNBS i n c o r p o r a t i o n on membrane p h o s p h o l i p i d s . As w i t h the p r o t e i n - a c t i v a t i o n energy e x p e r i m e n t s , t h e s e were under-t a k e n because of t h e o b s e r v e d p a r a l l e l between r a t e and phos-p h o l i p i d l a b e l l i n g p r o f i l e s . P h o s p h o l i p a s e A t r e a t m e n t de-c r e a s e d r a t e s o f TNBS i n c o r p o r a t i o n f o r a l l the c a t i o n s b u t 2 + 2 + most n o t i c e a b l y f o r the a l k a l i n e e a r t h c a t i o n s Mg , Ca , 2 + 2 + Sr , and Ba , w h i l e t r y p s i n i z a t i o n d i d not a f f e c t the r a t e s i n the p r e s ence o f these f o u r c a t i o n s ( F i g u r e 11, l o w e r ) . A l t h o u g h PC i s i n c o r p o r a t e d a t a c o n s i d e r a b l y h i g h e r r a t e t han TNBS (due to g r e a t e r p e r m e a t i o n o f PC), the i o n i c r a d i u s p r o f i l e s o f r a t e s o f i n c o r p o r a t i o n o f b o t h t h e s e probes were 2 + v e r y s i m i l a r , w i t h the e x c e p t i o n o f N i . The low r a t e o f 2 + PC i n c o r p o r a t i o n i n the p r e s e n c e of N i may be due to the v e r y h i g h a c t i v a t i o n energy term mentioned e a r l i e r . T h i n l a y e r c h r o m a t o g r a p h i c s t u d i e s o f i n d i v i d u a l p h o s p h o l i p i d s i n d i c a t e d t h a t the l a b e l l i n g o f p h o s p h a t i d y l e t h a n o l a m i n e p a r a l l e l e d the r a t e o f TNBS i n c o r p o r a t i o n i n whole membranes. T h i s was somewhat s u r p r i s i n g i n v i e w o f s t u d i e s on a r t i f i c i a l p h o s p h o l i p i d systems which have demonstrated t h a t d i v a l e n t c a t i o n s b i n d to a c i d i c p h o s p h o l i p i d s s uch as p h o s p h a t i d y l -s e r i n e (28, 32) b u t n o t to n e u t r a l p h o s p h o l i p i d s such as p h o s p h a t i d y l e t h a n o l a m i n e (12, 30). On the b a s i s o f t h e s e r e s u l t s , one might have p r e d i c e d t h a t p h o s p h a t i d y l s e r i n e l a b e l l i n g would be more l i k e l y to be a f f e c t e d by d i v a l e n t c a t i o n s than p h o s p h a t i d y l e t h a n o l a m i n e l a b e l l i n g . I t seems l i k e l y t h a t membrane s t r u c t u r a l components a r e c a p a b l e o f i n f l u e n c i n g the a b i l i t y o f membrane p h o s p h o l i p i d s t o i n t e r -a c t w i t h and be p e r t u r b e d by d i v a l e n t c a t i o n s . These con-s i d e r a t i o n s emphasize t h a t i t i s n o t r e a d i l y p o s s i b l e t o e x t r a p o l a t e m e a n i n g f u l l y from a r t i f i c i a l p h o s p h o l i p i d systems t o b i o l o g i c a l membranes. D i v a l e n t c a t i o n s thus appear a b l e t o a f f e c t b o t h p r o -t e i n and p h o s p h o l i p i d membrane components, a l t h o u g h t o d i f f e r e n t e x t e n t s . I t might be e x p e c t e d , t h e r e f o r e , t h a t the f u n c t i o n a l consequences o f d i v a l e n t c a t i o n a c t i o n would depend on the b a l a n c e between p r o t e i n and p h o s p h o l i p i d s t r u c t u r a l , a l t e r a t i o n s . R e f e r e n c e t o Table I i n d i c a t e s t h a t , on the b a s i s o f p h o s p h o l i p i d t o p r o t e i n l a b e l l i n g r a t i o s , the d i v a l e n t c a t i o n s f a l l i n t o two groups. The 2 + f i r s t group i s comprised o f the a l k a l i n e e a r t h c a t i o n s Mg , 2+ 2+ 2 + Ca , Sr , and Ba , w h i l e the r e m a i n i n g d i v a l e n t c a t i o n s 2+ 2+ 2 + Co , Mn , and N i form a second heterogeneous group. I n t e r e s t i n g l y , s i m i l a r g r o u p i n g s o f t h e s e c a t i o n s may be made on the b a s i s o f t h e i r a b i l i t y t o mod i f y f u n c t i o n a l p r o -p e r t i e s o f e x c i t a b l e t i s s u e s . The s l o w i n w a r d c u r r e n t i n mammalian m y o c a r d i a l f i b r e s w hich i s n o r m a l l y c a r r i e d by C a 2 + can a l s o be c a r r i e d by S r 2 + , B a 2 + , o r M g 2 + (17,71), w h i l e N i 2 + , C o 2 + , and Mn 2 + i n h i b i t t h i s c u r r e n t (17). I n 2+ 2+ + s q u i d g i a n t axons, Co and N i slow t h e K c u r r e n t k i n e t i c s 2 + w h i l e Ca has no e f f e c t , a f i n d i n g a t t r i b u t e d t o the g r e a t e r 2+ 2 + a b i l i t y o f N i and Co to b i n d t o p r o t e i n s (72). The 2+ 2+ 2 + e f f e c t s o f Mg , Sr , and Ba on p r e s y n a p t i c r e l e a s e o f a c e t y l c h o l i n e , b e l i e v e d t o be the r e s u l t of charge s c r e e n i n g , were n o t i c e a b l y d i f f e r e n t from the more c o m p l i c a t e d a c t i o n s 2+ 2 + o f d i v a l e n t c a t i o n s such as Mn and N i which can b i n d t o motor axons and t e r m i n a l s ( 7 3 ) . The a b i l i t y o f d i v a l e n t c a t i o n s to a l t e r t he c o n d u c t a n c e - v o l t a g e c h a r a c t e r i s t i c s o f nerve axons (74) has been e x p l a i n e d i n some systems as a c o m b i n a t i o n o f s c r e e n i n g and b i n d i n g ( 1 9 ) , w h i l e i n o t h e r systems,such as the c r a y f i s h axon, the l a c k o f s p e c i f i c i t y 2+ 2+ 2+ 2+ 2+ 2 + o f t h e e f f e c t s o f Mg , Ca , Sr , Ba , N i , and Co has been i n t e r p r e t e d as i n d i c a t i n g t h a t s c r e e n i n g a l o n e i s i n v o l v e d ( 18). In o r d e r to d e r i v e some i n f o r m a t i o n on the p o s s i b l e m o l e c u l a r b a s i s o f such d i v a l e n t c a t i o n e f f e c t s , i t i s r e l e -v a n t t o c o n s i d e r t h e r e s u l t s o f in_ y i t r o e x p e r i m e n t s u t i -l i z i n g a r t i f i c i a l membrane systems. Most o f these s t u d i e s have been conducted on a r t i f i c i a l p h o s p h o l i p i d systems and have i n d i c a t e d the f o r m a t i o n o f t r i d e n t a t e complexes com-posed o f two p h o s p h o l i p i d m o l e c u l e s w i t h the d i v a l e n t c a t i o n a c t i n g as a b r i d g e between the n e g a t i v e l y charged phosphate groups o f the p h o s p h o l i p i d s (21, 22, 33). The p o s s i b i l i t y o f extended complex f o r m a t i o n i n p h o s p h o l i p i d v e s i c l e s has su g g e s t e d t h a t d i v a l e n t c a t i o n s may i n d u c e a g g r e g a t i o n o f these v e s i c l e s by a p o l y m e r i z a t i o n type o f p r o c e s s (9, 23). A few s t u d i e s have a l s o shown t h a t p r o t e i n s can p a r t i c i p a t e i n such complex f o r m a t i o n , whereby the d i v a l e n t c a t i o n would a c t as a b r i d g e between n e g a t i v e s i t e s on b o t h the p r o t e i n and p h o s p h o l i p i d m o i e t i e s (24, 25). S t u d i e s w i t h the membrane enzymes a c e t y l c h o l i n e s t e r a s e and p - n i t r o p h e n y l p h o s p h a t a s e were u n d e r t a k e n i n o r d e r t o ex t e n d the e a r l i e r s t u d i e s of p r o t e i n s t r u c t u r a l p e r t u r b a -t i o n s u t i l i z i n g TNBS l a b e l l i n g . I t was hoped t h a t , i n ad-d i t i o n t o e f f e c t s on p r o t e i n s , some i n f o r m a t i o n on the v e c t o r i a l a s p e c t s o f d i v a l e n t c a t i o n a c t i o n might a l s o be d e r i v e d . These experiments a l s o r e p r e s e n t e d an i n i t i a l a pproach t o t h e s t u d y o f f u n c t i o n a l conseuences o f membrane-c a t i o n i n t e r a c t i o n s . The e x p e r i m e n t s w i t h the membrane en-zyme a c e t y l c h o l i n e s t e r a s e , an enzyme a t the o u t e r membrane s u r f a c e ( 5 0 ) , tended to emphasize the p r e v i o u s l y n o t e d 2+ 2 + d i f f e r e n c e s between N i and Co as compared t o the o t h e r d i v a l e n t c a t i o n s w i t h r e g a r d t o e f f e c t s on p r o t e i n . These e x p e r i m e n t s a l s o s u g g e s t e d t h a t e i t h e r t h e e x t e r i o r a s p e c t of the membrane i s n o t a s i t e o f major c o n f o r m a t i o n a l changes, s i n c e e f f e c t s were g e n e r a l l y s l i g h t , o r t h a t the changes were such t h a t they d i d not markedly a f f e c t the c h o l i n e s t e r a s e enzyme ( F i g u r e 1 6 ) . The enzyme p - n i t r o p h e n y l p h o s p h a t a s e , which i t was hoped would g i v e a b e t t e r i n d i c a t i o n o f t h e v e c t o r i a l c h a r a c t e r i s -t i c s o f cation-membrane i n t e r a c t i o n s i n c e i t has i o n - b i n d i n g s i t e s a t b o t h the i n n e r and o u t e r s u r f a c e of the membrane (5 2 ) , p r o v e d to be d i s a p p o i n t i n g i n t h i s r e s p e c t . However, 2 + the a b i l i t y o f Mn to s t i m u l a t e and o f the o t h e r d i v a l e n t c a t i o n s t o i n h i b i t t he b a s a l a c t i v i t y o f p - n i t r o p h e n y l p h o s -p h a t a s e ( F i g u r e 18) suggested t h a t the c a t i o n s were c a p a b l e of p e r t u r b i n g the i n n e r membrane s u r f a c e , from which the b a s a l a c t i v i t y o r i g i n a t e s . F u r t h e r m o r e , the g r e a t e r e f f e c t on the K + - s t i m u l a t e d component o f t h e enzyme ( F i g u r e 18) as compared to the b a s a l a c t i v i t y may be i n t e r p r e t e d as o c c u r r i n g as a r e s u l t o f d i v a l e n t c a t i o n - i n d u c e d s t r u c t u r a l a l t e r a t i o n s a t the o u t e r membrane s u r f a c e where K + e x e r t s i t s a c t i o n . I t may be n o t e d t h a t a l l the d i v a l e n t c a t i o n s s t u d i e d were c a p a b l e o f i n t e r a c t i n g w i t h the c r i t i c a l s i t e on the i n n e r 2 + 2 + membrane s u r f a c e b u t o n l y Mg and Mn were a b l e t o i n d u c e a c a t a l y t i c a l l y a c t i v e c o n f o r m a t i o n i n the enzyme on combina-2 + t i o n w i t h t h i s s i t e . S i m i l a r i t y i n b e h a v i o u r o f Mg and Mn^ + has a l s o been o b s e r v e d i n the s q u i d g i a n t axon, where i n h i b i t i o n o f t r a n s m i t t e r r e l e a s e , r a t h e r t h a n the enhanced 2 + r e l e a s e produced by o t h e r d i v a l e n t c a t i o n s (such as Ca o r S r ^ + ) , has been o b s e r v e d (14). S o n i c a t i o n s t u d i e s on e r y t h r o c y t e membranes i n the p r e -sence of d i v a l e n t c a t i o n s were u n d e r t a k e n as an a l t e r n a t i v e method o f a n a l y z i n g membrane-cation i n t e r a c t i o n , one w h i c h would be independent of the i n c o r p o r a t i o n o f a c o v a l e n t l y bound probe and would not i t s e l f g i v e r i s e to s t r u c t u r a l changes i n t h e membrane. The e f f e c t s o f d i v a l e n t c a t i o n s on membrane t u r b i d i t y f o l l o w i n g s o n i c a t i o n showed a d i s t i n c t s i m i l a r i t y to t h e i r s t i m u l a t o r y e f f e c t s on TNBS i n c o r p o r a -t i o n i n t o membranes ( F i g u r e 20). When s i a l i c a c i d i s r e -moved e n z y m a t i c a l l y by n e u r a m i n i d a s e , a g g r e g a t i o n o f e r y t h r o c y t e s o c c u r s ( F i g u r e 22, i n s e t ) , and t h e d i v a l e n t 2+ 2+ 2 + c a t i o n s Co , Mi , and Mn l o s e t h e i r a b i l i t y to p r o t e c t a g a i n s t s o n i c a t i o n , s u g g e s t i n g t h a t the s i a l i c a c i d c a r b o x y l g r o u p s a r e p r o b a b l y a n i m p o r t a n t s i t e o f a c t i o n o f t h e s e c a t i o n s i n m e d i a t i n g t h i s p r o t e c t i o n ( F i g u r e 2 2 ) . The l a c k o f e f f e c t o f n e u r a m i n i d a s e t r e a t m e n t on t h e p r o t e c t i v e a c t i o n o f a l k a l i n e e a r t h c a t i o n s r e i n f o r c e s p r e v i o u s o b s e r v a t i o n s t h a t t h e s e c a t i o n s f o r m a d i s t i n c t g r o u p w i t h r e g a r d t o t h e i r i n t e r a c t i o n w i t h membranes. P h o s p h o l i p a s e A t r e a t m e n t a nd t r y p s i n i z a t i o n , w h i c h were u n d e r t a k e n t o e x a m i n e t h e r o l e o f l i p i d s and p r o t e i n s i n c a t i o n e f f e c t s , w e r e f o u n d t o a f f e c t t h e a b i l i t y o f t h e c a t i o n s t o p r o t e c t t h e membranes f r o m s o n i c a t i o n , a l t h o u g h t o d i f f e r e n t e x t e n t s f o r t h e v a r i o u s c a t i o n s . T h e s e f i n d i n g s a r e c o n s i s t e n t w i t h t h e i d e a t h a t b o t h p r o t e i n s a n d p h o s p h o l i p i d s a r e i m p o r t a n t i n d e t e r m i n i n g membrane a l t e r a t i o n s i n d u c e d b y c a t i o n s , p e r h a p s b y means o f b r i d g i n g o f membrane s t r u c t u r a l c o m p o n e n t s b y d i v a l e n t c a t i o n s s o t h a t t h e membrane i s more r e s i s t e n t t o d i s i n t e -g r a t i o n . T h e s e e x p e r i m e n t s a l s o f u r t h e r c o n f i r m t h e s i m i -l a r i t y o f a c t i o n o f t h e a l k a l i n e e a r t h c a t i o n s as a g r o u p and p r o v i d e f u r t h e r e v i d e n c e t h a t t h e membrane p e r t u r b a -t i o n a l e f f e c t s o f e a c h d i v a l e n t c a t i o n i s d e t e r m i n e d b y i t s r e l a t i v e a b i l i t y t o i n t e r a c t w i t h a n d p e r t u r b p r o t e i n a n d p h o s p h o l i p i d membrane c o m p o n e n t s . E x p e r i m e n t s o n t h e r e s p o n s e s o f i n t a c t e r y t h r o c y t e s t o a h y p o t o n i c c h a l l e n g e i n t h e p r e s e n c e o f v a r i o u s d i v a l e n t c a t i o n s w e r e u n d e r t a k e n i n o r d e r t o compare t h e i r p r o p e r t i e s o n i n t a c t c e l l s as o p p o s e d t o e r y t h r o c y t e g h o s t s a nd a s a p o s s i b l e a p p r o a c h t o f u n c t i o n a l a l t e r a t i o n s i n d u c e d b y d i v a l e n t c a t i o n s . H e m o l y s i s was c h o s e n b e c a u s e o f t h e p r o -posed r e l a t i o n s h i p between the e f f e c t s o f a n t i h e m o l y t i c agents i n r e d b l o o d c e l l s and e x c i t a b l e t i s s u e s (75, 76). The r e s u l t s o f these experiments ( F i g u r e 1 9 ) , showed a s u r -p r i s i n g degree of s i m i l a r i t y t o the a c t i v a t i o n energy o f TNBS i n c o r p o r a t i o n ( F i g u r e 6) and the maximal TNBS i n c o r -p o r a t i o n i n t o p r o t e i n ( F i g u r e 1 3 ) . The c o r r e l a t i o n ( F i g u r e 19 and Table I ) between a n t i h e m o l y t i c p r o p e r t i e s o f the a l k a l i n e e a r t h c a t i o n s and t h e i r p h o s p h o l i p i d to p r o t e i n l a b e l l i n g r a t i o s i s c o n s i s t e n t w i t h a common s i t e and/or mechanism o f a c t i o n o f the c a t i o n s w i t h the membrane, p o s s i b l y i n v o l v i n g s c r e e n i n g . The more complex e f f e c t s o f Co^ +, N i ^ + , and Mn^ + may be due to t h e i r g r e a t e r a b i l i t y t o i n t e r a c t w i t h membrane p r o t e i n s . The s i m i l a r i t i e s between i n t a c t e r y t h r o c y t e s and e r y t h r o c y t e g h o s t s may be i n d i c a t i v e o f the i m p o r t a n c e o f the e x t e r n a l membrane s u r f a c e i n d i v a -l e n t c a t i o n i n t e r a c t i o n s , c o n s i s t e n t w i t h t h e above men-t i o n e d s t u d i e s on p - n i t r o p h e n y l p h o s p h a t a s e . The p r e l i m i n a r y s t u d i e s on membranes from e x c i t a b l e ( b o v i n e , s y n a p t i c v e s i c l e s ) and n o n - e x c i t a b l e ( r a t l i v e r m i crosomal membranes) t i s s u e s showed b o t h s i m i l a r i t i e s and d i f f e r e n c e s w i t h r e s p e c t t o e r y t h r o c y t e g h o s t s . The s i m i -l a r i t i e s between the membrane systems s u g g e s t t h a t f u n d a -mental i n f o r m a t i o n on the a c t i o n o f d i v a l e n t c a t i o n s d e r i v e d from s t u d i e s on r e a d i l y a v a i l a b l e and w e l l c h a r a c t e r i z e d membrane systems such as the e r y t h r o c y t e g h o s t may be e x t r a p o l a t e d t o o t h e r systems and, i n p a r t i c u l a r , may p r o v i d e some i n s i g h t i n t o the mechanisms whereby d i v a l e n t c a t i o n s a l t e r the e l e c t r i c a l p r o p e r t i e s o f e x c i t a b l e t i s s u e s . The q u a n t i t a t i v e v a r i a t i o n s o b s e r v e d a re p r o b a b l y due to d i f f e r -ences i n p r o t e i n and p h o s p h o l i p i d c o m p o s i t i o n o f the membrane systems examined. Experiments a re c u r r e n t l y under way to e x t e n d t h e s e s t u d i e s o f d i v a l e n t c a t i o n a c t i o n on membrane systems by u t i l i z i n g c hromophoric b i f u n c t i o n a l c r o s s - l i n k i n g agents i n o r d e r to g a i n a more d e t a i l e d u n d e r s t a n d i n g o f r e s u l t i n g s t r u c t u r a l p e r t u r b a t i o n s i n terms o f a l t e r a t i o n s i n p r o t e i n -p r o t e i n , l i p i d - l i p i d , and l i p i d - p r o t e i n i n t e r a c t i o n s . Ex-p e r i m e n t s u t i l i z i n g the f o r e g o i n g approach to a n a l y z e the m o l e c u l a r p e r t u r b a t i o n s i n d u c e d by drugs i n membranes d e r i v e d from e l e c t r i c a l l y e x c i t a b l e t i s s u e s a r e c u r r e n t l y b e i n g u n d e r t a k e n i n our l a b o r a t o r y . I n a d d i t i o n , i t was o b s e r v e d t h a t TNBS i n c o r p o r a t i o n i n the p r e s e n c e of d i v a l e n t c a t i o n s was a p p a r e n t l y much more s e n s i t i v e t o c o n f i g u r a t i o n a l a l t e r a -t i o n s i n membrane p r o t e i n s and p h o s p h o l i p i d s t h a n , i n the absence o f these c a t i o n s , based on r e s u l t s o b t a i n e d u s i n g membranes p r e t r e a t e d w i t h p h o s p h o l i p a s e o r p r o t e o l y t i c en-zymes ( F i g u r e 1 1 ) . These f i n d i n g s s u g g e s t e d t h a t group-s p e c i f i c c h e m i c a l probes such as TNBS c o u l d be used i n the p r e s e n c e o f d i v a l e n t c a t i o n s as a more s e n s i t i v e method o f d e t e c t i n g and a n a l y z i n g membrane l e s i o n s , e i t h e r t h o s e i n -duced by p h a r m a c o l o g i c a l agents o r o c c u r r i n g n a t u r a l l y as a r e s u l t o f some p a t h o l o g i c a l p r o c e s s . With r e s p e c t t o t h i s , s t u d i e s are c u r r e n t l y i n p r o g r e s s i n our l a b o r a t o r y r e g a r d i n g h e p a t o t o x i n - i n d u c e d damage to l i v e r m icrosomal membranes and erythrocyte membrane alterations occurring i n human Duchenne muscular dystrophy. 93 BIBLIOGRAPHY 1. R e y n o l d s , J.A. , Fed. P r o c . _32 2034-2038 (1973) 2. B u r g e r , S.P., F u j i i , T., and Hanahan, D.J., B i o c h e m i s t r y 1_ 3682-3700 (1968) 3. R a z i n , S., B i o c h i m . B i o p h y s . A c t a 265 241-296 (1972) 4. Manery, J . F . , F e d . P r o c . 25 1804-1810 (1966) 5. B u r t , D.H. and Green, J.W., B i o c h i m . B i o p h y s . A c t a 225 46-55 (1971) 6. H o d g k i n , A.L., P r o c . Roy. Soc. London S e r i e s B 148 1-37 (1958) 7. T o b i a s , J.M., A g i n , D.P., and P a w l o w s k i , R., C i r c u l a t i o n 26. 1145-1150 (1962) 8. P o s t e , G. and A l l i s o n , A.C., B i o c h i m . B i o p h y s . A c t a 300 421-465 (1973) 9. Lansman, J . and Haynes, D.H., B i o c h i m . B i o p h y s . A c t a 394 335-347 (1975) 10. J a n , K.M. and C h i e n , S., J . Gen. P h y s i o l . 61 655-668 (1973) 11. Maeda, T. and O h n i s h i , S . I . , Biochem. B i o p h y s . Res. Comm. 60 1509-1516 (1974) 12. O h n i s h i , S . I . and I t o , T. , B i o c h e m i s t r y 1_3 881-887 (1974) 13. Edwards, W., P h i l l i p s , J.H., and M o r r i s , S . J . , B i o c h i m . B i o p h y s . A c t a 356 164-173 (1973) 14. M i l e d i , R., P r o c . Roy. Soc. London S e r i e s B 18 3 421-425 (1973) 15. M e i r i , U. and Rahamimoff, R., J . P h y s i o l . 215 709-726 (1971) 16. H a g i w a r a , S., Fukuda, J . , and E a t o n , D.C., J . Gen. P h y s i o l . 63_ 564-578 (1974) 17. K o h l h a r d t , M. , H e r d e y , A. , and K i i b l e r , M. , P f l u e g e r s A r c h . 344 149-158 (1973) 18. D ' A r r i g o , J . S . , J . P h y s i o l . 231 117-128 (1973) 94 19. M c L a u g h l i n , S.G.A., Szabo, G., and Eisenman, G., J . Gen. P h y s i o l . 58_ 667-687 (1971) 20. Gent, W.L.G., Trounce, J.R., and Wal s e r , M., A r c h . Biochem. B i o p h y s . 105 582-589 (1964) 21. B l a u s t e i n , M.P., Bioc h i m . B i o p h y s . A c t a 135 653-668 (1967) 22. Shah, D.O. and Schulman, J.H. , J . L i p i d Res. 8_ 227-233 (1967) 23. P a p a h a d j o p o u l o s , D., B i o c h i m . B i o p h y s . A c t a 163 240-254 (1968) 24. B u l k i n , B.J. and Hauser, R., Bi o c h i m . Biophys. A c t a 326 289-292 (1973) 25. J u l i a n o , R.L., K i m e l b e r g , H.K., and Papa h a d j o p o u l o s , D. , Bioch i m . B i o p h y s . A c t a 241 894-905 (1971) 26. E h r s t r o m , M. , Goran E r i k s s o n , L.E., I s r a e l a c h v i l i , J . , and Ehrenberg, A., Biochem. Biophy s . Res. Comm. 55_ 396-402 (1973) 27. Hauser, H., P h i l l i p s , M.C., and B a r r a t t , M.D., Bi o c h i m . B i o p h y s . A c t a 413 341-353 (1975) 28. S c h n e p e l , G.H., Hegner, D., and Schummer, U., B i o c h i m . B i o p h y s . A c t a 2&1 67-74 (1974) 29. O h n i s h i , S.I. and I t o , T., Biochem. B i o p h y s . Res. Comm. 51 132-138 (1973) 30. I t o , T. and O h n i s h i , S . I . , B i o c h i m . B i o p h y s . A c t a 352 29-37 (1974) 31. T r a u b l e , H. and E i b l , H. , Proc. Nat. Acad. S c i . 71_ 214-219 (1974) 32. J a c o b s o n , K. and Pap a h a d j o p o u l o s , D. , B i o c h e m i s t r y 1_4 152-161 (1975) 33. V e r k l e i j , A . J . , de K r u y f f , B., V e r v e r g a e r t , P.H.J.Th., Tocanne, J . F . , and Van Deenen, L.L.M., Bio c h i m . B i o p h y s . A c t a 339 432-437 (1974) 34. R u b a l c a v a , B., M a r t i n e z de Munoz, D., and G i t l e r , C., B i o c h e m i s t r y 8 2742-2747 (1969) 35. V a n d e r k o b i , J . and M a r t o n o s i , A., A r c h . Biochem. B i o p h y s . 133 153Q163 (1969) 36. F l a n a g a n , M. T. and Hes k e t h , T.R. , B i o c h i m . B i o p h y s . A c t a 298 535-545 (1973) 37. Godin, D.V. and Wan Ng, T., Mol. Pharmacol. 8 426-437 (1972) 38. Godin, D. V. and Wan Ng, T. , Mol. Pharmacol. 9_ 802-819 (1973) 39. Godin, D. V. and S c h r i e r , S.L. , B i o c h e m i s t r y 9_ 4068-4077 (1970) 40. C o r b e t t , L., C h r i s t i a n , S.T., M o n t i , J.A., and M c C l a i n , L.D. , Res. Comm. i n Chem. P a t h o l , and Pharmacol. 8_ 607-614 (1974) 41. Kamath, S.A. and Ananth Narayan, K. , A n a l . Biochem. 48_ 53-61 (1972) 42. Weihing, R.R., M a n g a n i e l l o , V.C., C h i u , R., and P h i l l i p s , A.H., B i o c h e m i s t r y 11 3128-3135 (1972) 43. Lowry, O.H., Rosebrough, N.J., F a r r , A.L., and R a n d a l l , R.J., J . B i o l . Chem. 193 265-275 (1951) 44. B a r t l e t t , G.R., J . B i o l . Chem. 234 466-468 (1959) 45. Zak, B. , Dickenman, R.C, White, F.G. , B u r n e t t , H. , and Cherney, P.J., Amer. J . C l i n . P a t h o l . 24 1307-1315 (1954) 46. Warren, L., J . B i o l . Chem. 234 1971-1975 (1959) 47. E l l m a n , G.L. , A r c h . Biochem. B i o p h y s . 8_2 70-77 (1959) 48. Hunt, J.P., "Metal Ions i n Aqueous S o l u t i o n " , W.A. Benjamin I n c . , N. Y., Amsterdam (1963) 49. C o t t o n , F.A. and W i l k i n s o n , G., "Advanced I n o r g a n i c C h e m i s t r y " 2nd e d i t i o n , I n t e r s c i e n c e P u b l i s h e r s , N.Y., London (1966) 50. H e l l e r , M. and Hanahan, D.J., Biochim. B i o p h y s . A c t a 255 251-272 (1972) 51. Ahmed, K. and Judah, J.D. , Bi o c h i m . Biophy. A c t a 9_3 603-613 (1964) 52. Skou, J.C., B u t l e r , K.W., and Hansen, O., Biochim. B i o p h y s . A c t a 241 443-461 (1971) 53. Weed, R.I. and Reed, C.F., Amer. J . Med. 41_ 681-698 (1966) 54. Zimmer, G. and S c h i r m e r , H., Bio c h i m . Biophys. A c t a 345 314-320 (1974) 96 55. M a c h t i g e r , N.A. and Fox, C.F., Ann. Rev. Biochem. 42_ 575-600 (1973) 56. S u t h e r l a n d , E.W., R a i l , T.W., and Menon, T., J . B i o l . Chem. 2_37 1220-1227 (1962) 57. Bronk, J.R. and K i e l l e y , W.W., B i o c h i m . B i o p h y s . A c t a 24 440-441 (1957) 58. A t k i n s o n , A. and Lowe, A.G., B i o c h i m . B i o p h y s . A c t a 266 103-115 (1972) 59. Coleman, R., B i o c h i m . B i o p h y s . A c t a 300 1-30 (1973) 60. P h i l l i p s , M.C., F i n e r , E.G., and H a u s e r , H., B i o c h i m . B i o p h y s . A c t a 290 397-402 (1970) 61. J e n d r a s i a k , G.L. and H a s t y , J.H., B i o c h i m . B i o p h y s . A c t a 33J_ 79-91 (1974) 62. R i g a u d , J . L . , Lange, Y., Gary-Bobo, G.M., Samson, A., and P t a k , M. , Biochem. B i o p h y s . Res. Comm. 5_0 59-65 (1973) 63. M i s i o r o w s k i , R.L. and W e l l s , M. A. , B i o c h e m i s t r y 1_2 967-975 (1973) 64. Rand, R.P. and S e n g u p t a , S., B i o c h i m . B i o p h y s . A c t a 255 484-492 (1972) 65. Meryman, H.T., i n "Biomembranes" V o l . 3, ed. by F. K r e u z e r and J.F.G. S i e g e r s , Plenum P r e s s , N.Y., London (1972) p.341 66. G u l , S. and S m i t h , A.D., B i o c h i m . B i o p h y s . A c t a 288 237-240 (1972) 67. S i m p k i n s , H. , Tay, S. , and Panko, E. , B i o c h e m i s t r y 1_0 3579-3585 (1971) 68. B e s n u e l l e , P. i n "The Enzymes" V o l . 4, ed. by P.D. B o y e r , H. L a r d y and K. Myrback, A c a d e m i c P r e s s , N.Y., London (1960) p.119 69. E y l a r , E.H. , M a d o f f , M.A., B r o d y , O.V., and O n c l e y , J . L . , J . B i o l . Chem. _237 1992-2000 (1962) 70. J a c k s o n , L . J . and Seaman, G.V.F., B i o c h e m i s t r y L l 44-49 (1972) 71. K o h l h a r d t , M., H a a s t e r t , H.P., and K r a u s e , H., P f l u e g e r s A r c h . 342 125-136 (1973) 97 72. B e g e n i s i c h , T. and L y n c h , C. , J . Gen. P h y s i o l . 6_3 675-689 (1974) 73. M u l l e r , R. U. ,and F i n k e l s t e i n , A., P r o c . Nat. Acad. S c i . 71_ 923-926 (1974) 74. F r a n k e n h a e u s e r , B. and H o d g k i n , A.L., J . P h y s i o l . 137 218-244 (1957) 75. R o t h , S. and Seeman, P., N a t u r e (New B i o l . ) 231 284-285 (1971) 76. Seeman, P., P h a r m a c o l . Rev. 24 583-655 (1972) 

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